Methods And Compositions Related To Thioesterase Enzymes

ABSTRACT

The present invention relates to novel mutant thioesterase enzymes and naturally-occurring equivalents thereof, compositions made from such enzymes and uses of thioesterase enzymes. In particular, the present invention provides mutant thioesterase enzymes that have altered properties, for example, altered substrate specificity, altered activity, altered selectivity, and/or altered proportional yields in the product mixtures. The present invention also provides polynucleotides encoding such mutant thioesterase enzymes, and vectors and host cells comprising such polynucleotides. The invention further provides for novel uses of thioesterases in the production of various fatty acid derivatives, which are useful as, or as components of, industrial chemicals and fuels.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/958,229, filed on Apr. 20, 2018, which is a continuation of U.S.application Ser. No. 15/439,053, filed on Feb. 22, 2017, which is acontinuation of U.S. application Ser. No. 14/826,657, filed on Aug. 14,2015, which is a divisional of U.S. application Ser. No. 12/645,497,filed on Dec. 23, 2009, which claims the benefit of U.S. ProvisionalApplication No. 61/140,600, filed Dec. 23, 2008, all of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel thioesterase compositions, novelrecombinant host cells comprising thioesterases, novel methods ofproduction of fatty acid derivatives, and fatty acid derivativesproduced thereby and uses thereof. One particular aspect of the presentinvention relates to the production of industrial chemicals and fuels.

BACKGROUND OF THE INVENTION

Developments in technology have been accompanied by an increasedreliance on fuel and industrial chemicals from petrochemical sources.Such fuel sources are becoming increasingly limited and difficult toacquire. With the burning of fossil fuels taking place at anunprecedented rate, it is likely that the world's demand for fuel andpetrochemical derived chemicals will soon outweigh current supplies.

As a result, efforts have been directed toward harnessing sources ofrenewable energy, such as sunlight, water, wind, and biomass. The use ofbiomass to produce new sources of fuel and chemicals which are notderived from petroleum sources (e.g., biofuel) has emerged as onealternative option.

Biofuel is a biodegradable, clean-burning combustible fuel which can becomprised of alkanes and/or esters. An exemplary biofuel is biodiesel.Biodiesel can be used in most internal combustion diesel engines ineither a pure form, which is referred to as “neat” biodiesel, or as amixture in any concentration with regular petroleum diesel or otherbiodiesels.

Biodiesel offers a number of beneficial properties compared topetroleum-based diesel, including reduced emissions (e.g., carbonmonoxide, sulphur, aromatic hydrocarbons, soot particles, etc.) duringcombustion. Biodiesel also maintains a balanced carbon dioxide cyclebecause it is based on renewable biological materials. Biodiesel istypically completely biodegradable, and has good safety profile due toits relative high flash point and low flammability. Furthermore,biodiesel provides good lubrication properties, thereby reducing wearand tear on engines.

Current methods of making biodiesel involve transesterification oftriacylglycerides from vegetable oil feedstocks, such as from rapeseedin Europe, from soybean in North America, and from palm oil in SouthEast Asia. Industrial-scale biodiesel production is thus geographicallyand seasonally restricted to areas where vegetable oil feedstocks areproduced. The transesterification process leads to a mixture of fattyesters which can be used as biodiesel, but also to an undesirablebyproduct, glycerin. To be usable as biodiesel, the fatty esters must befurther purified from the heterogeneous product. This increases costsand the amount of energy required for fatty ester production and,ultimately, biodiesel production as well. Furthermore, vegetable oilfeedstocks are inefficient sources of energy because they requireextensive acreage for cultivation. For example, the yield of biodieselfrom rapeseed is only 1300 L/hectare because only the seed oil is usedfor biodiesel production, and not the rest of the rapeseed biomass.Additionally, cultivating some vegetable oil feedstocks, such asrapeseed and soybean, requires frequent crop rotation to preventnutrient depletion of the land.

PCT Publication No. WO 2007/136762 discloses recombinant microorganismsthat are capable of synthesizing products derived from the fatty acidsynthetic pathway, including, inter alia, fatty acid esters and fattyalcohols. In particular, certain fatty acid derivatives are describedhaving defined carbon chain length, branching and saturation levels. The'762 publication describes recombinant cells that utilize endogenousoverexpression or heterologous expression of thioesterase proteins inthe production of fatty acid derivatives.

PCT Publication No. WO 2008/119082 discloses genetically engineeredcells and microorganisms that produce products from the fatty acidbiosynthetic pathway, including, inter alia, fatty acid esters and fattyalcohols. The '082 publication describes recombinant cells that utilizeoverexpression of acyl-CoA synthetase enzymes to more efficientlyproduce fatty acid derivatives.

U.S. Pat. No. 5,955,329 discloses genetically engineered plant acyl-ACPthioesterase proteins having altered substrate specificity. Inparticular, the '329 patent discloses producing engineered plantacyl-ACP thioesterases, wherein the engineered plant acyl-ACPthioesterases demonstrate altered substrate specificity with respect tothe acyl-ACP substrates hydrolyzed by the plant thioesterases ascompared to the native acyl-ACP thioesterase.

While the prior art discloses certain useful disclosures regarding theproduction of certain fatty acid derivatives, a need exists in the fieldfor improved methods and processes for more efficient and economicalproduction of such fatty acid derivatives, and also for technologyfacilitating the production of compositions that have altered productspecifications. As a specific example, a need exists for the productionof fatty acid compositions having pre-designed, or “tailored,”specifications and properties for particular applications such as fuels,detergents, lubricants, industrial precursor molecule and other valuableapplications of fatty acid derivatives.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide useful mutant andnaturally-occurring thioesterase enzymes, polynucleotides encoding theseenzymes, vectors comprising polynucleotides encoding the usefulthioesterase enzymes, recombinant host cells comprising mutatedendogenous thioesterase enzymes, recombinant host cells transformed withthe vectors, recombinant host cells having polynucleotides encodinguseful thioesterase enzymes chromosomally integrated therein,thioesterases produced by the host cells, fatty acid derivativecompositions (such as industrial chemicals and biofuels) produced invitro and/or in vivo, methods for producing fatty acid derivativecompositions in vitro and/or in vivo, and methods of using the producedfatty acid derivative compositions.

It is an object of the present invention to provide methods of producingfatty acid derivative compositions through microbial fermentations thathave predetermined product profiles with regard to carbon chain lengthsand proportional yields. These compositions are well suited forapplications in the fuel and chemical industries because theirproperties can be tailored to the particular applications for which theyare intended. For example, it is possible to tailor a fatty esterproduct, according to the methods described herein, such that it can beused as an automobile fuel, and/or to design a composition to have, forexample, improved fuel characteristics such as cloud point, lubricity,cetane number, kinematic viscosity, acid number, boiling point,oxidative stability, cold filter-plugging point, impurity profile,sulfated ash level, and/or flash point. Similarly, it is possible toproduce industrial chemicals in accordance with the methods describedherein that can replace current chemicals sourced from petroleum, andthat are tailored to particular applications, for example, to producefatty alcohols that are optimally suited for use as surfactants and/ordetergents.

It is an object of the invention to provide for alternative methods ofmaking fatty esters without the presence of (or in the absence of) anester synthase. This method is energetically more favorable than theheretofore disclosed methods for producing fatty ester compositionsthrough microbial fermentation processes, which required at least both athioesterase enzyme and an ester synthase enzyme. As such, the novelthioesterases of the invention provide further advantages.

In one embodiment of the invention, mutant thioesterases (ornaturally-occurring equivalents thereof) are provided that derive from aprecursor thioesterase, wherein each of the mutants (or thenaturally-occurring equivalents) has at least one altered property invitro and/or in vivo, as compared to the properties of the precursorthioesterase. The altered property can be, for example, a biophysicalproperty such as thermal stability (melting point T_(m)); solvent,solute, and/or oxidative stability; lipophilicity; hydrophilicity;quaternary structure; dipole moment; and/or isoelectric point. Thealtered property can also be, for example, a biochemical property suchas pH optimum, temperature optimum, and/or ionic strength optimum. Thealtered property can further be, for example, an enzyme catalyticparameter such as product distribution (including, for example, a higheror lower percentage or proportional yield for a particular product vs.other products in the product mixture), specific activity, substratepreference, substrate affinity, substrate inhibition, product affinity,turnover rate or catalytic rate, product inhibition, kinetic mechanism,K_(M), k_(cat), k_(cat)/K_(m), and/or V_(Max). The altered property canadditionally be, for example, an increase or a decrease in activity or achanged preference for alcoholysis vs. hydrolysis, acyl-CoA vs.acyl-acyl carrier protein substrates, ester vs. thioester substrates,saturated vs. unsaturated substrates, straight-chain vs. branchedsubstrates; changes in positions of unsaturations, ranges of cetanenumbers, or specific carbon chain lengths, branched substrates, positionof branching, hydroxy-acyl substrates, keto-acyl substrates; and/orproducts with a changed range of or specific cetane numbers, octanerating, oxidative stability, lubricity, flash point, viscosity, boilingpoint, melting point, pour point, cloud point, cold filter pluggingpoint, cold flow characteristics, aromaticity, and/or iodine number.Altered properties can also include, for example, a decrease in activityor an attenuation of ester hydrolysis, such that the hydrolysis ofdesired product molecules is reduced or eliminated. Altered propertiescan further include, for example, a decrease in the protein's toxicityto the cell and/or a change in the protein's expression level in thecell, as compared to the precursor protein's toxicity to and/orexpression level in the same cell. In an exemplary embodiment, analtered property can include a change in the ability to catalyze thesynthesis of fatty acyl derivatives directly or indirectly in vivo or invitro. In another exemplary embodiment, an altered property is theimprovement or increase of in vitro and/or in vivo yield or proportionalyield of a particularly desirable fatty acid derivative.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is derived from a precursorthioesterase. In a particular embodiment of the invention, the precursorthioesterase is a naturally-occurring thioesterase, a previouslymodified thioesterase, or a synthetic thioesterase.

In one embodiment of the invention, the mutant thioesterase (or anaturally-occurring equivalent thereof) is derived from a precursorthioesterase that is a naturally-occurring thioesterase. Thenaturally-occurring precursor thioesterase can be obtained from, forexample, a plant, animal, bacterial, fungal, yeast, or other microbialsources. The mutant thioesterase (or a naturally-occurring equivalentthereof) can be derived from a precursor thioesterase from Acidovorax,Acinetobacter, Aeromonas, Alcanivorax, Aliivibrio, Alkalilimnicola,Alteromonadales, Alteromonas, Aurantimonas, Azoarcus, Azorhizobium,Azotobacter, Beggiatoa, Beijerinckia, Bordetella, Bradyrhizobium,Burkholderia, Caulobacter, Cellvibrio, Chromobacterium, Citrobacter,Comamonas, Cupriavidus, Dechloromonas, Delftia, Desulfovibrio,Enterobacter, Erwinia, Escherichia, Geobacter, Hahella, Halorhodospira,Herminiimonas, Idiomarina, Janthinobacterium, Klebsiella, Leptospira,Leptothrix, Limnobacter, Magnetospirillum, Marinobacter, Marinomonas,Methylibium, Methylobacillus, Methylobacterium, Methylocella,Methylococcus, Moritella, Nitrobacter, Nitrococcus, Nitrosomonas,Nitrosospira, Oceanospirillum, Oligotropha, Pectobacterium,Photobacterium, Photorhabdus, Polaromonas, Proteus, Providencia,Pseudoalteromonas, Pseudomonas, Psychromonas, Ralstonia, Reinekea,Rhodobacterales, Rhodoferax, Rhodopseudomonas, Rhodospirillum,Saccharophagus, Salmonella, Serratia, Shewanella, Shigella,Stenotrophomonas, Streptococcus, Thauera, Thioalkalivibrio,Thiobacillus, Vibrio, Xanthomonas, or Yersinia.

In a particular embodiment, the precursor thioesterase of the inventioncan be derived from any one of Acidovorax avenae subsp. citrulliAAC00-1, Acidovorax sp. JS42, Acinetobacter baumannii ACICU,Acinetobacter baumannii ATCC 17978, Aeromonas hydrophila subsp.Hydrophila ATCC 7966, Aeromonas salmonicida subsp. salmonicida A449,Alcanivorax borkumensis SK2, Alcanivorax sp. DG881, Aliivibriosalmonicida LFI1238, Alkalilimnicola ehrlichei MLHE-1, alphaproteobacterium HTCC2255, Alteromonadales bacterium TW-7, Alteromonasmacleodii deep ecotype, Aurantimonas sp. SI85-9A1, Azoarcus sp. BH72,Azorhizobium caulinodans ORS 571, Azotobacter vinelandii AvOP, Beggiatoasp. PS, Beijerinckia indica subsp. indica ATCC 9039, Bordetella avium197N, Bordetella bronchiseptica RB50, Bordetella parapertussis 12822,Bordetella pertussis Tohama I, Bordetella petrii DSM 12804,Bradyrhizobium sp. BTAi1, Bradyrhizobium sp. ORS278, Burkholderiaambifaria AMMD, Burkholderia ambifaria IOP40-10, Burkholderia ambifariaMC40-6, Burkholderia ambifaria MEX-5, Burkholderia cenocepacia AU 1054,Burkholderia cenocepacia HI2424, Burkholderia cenocepacia J2315,Burkholderia cenocepacia MCO-3, Burkholderia cenocepacia PC184,Burkholderia dolosa AU0158, Burkholderia graminis C4D1M, Burkholderiamallei ATCC 23344, Burkholderia mallei GB8 horse 4, Burkholderia malleiNCTC 10229, Burkholderia multivorans ATCC 17616, Burkholderiaoklahomensis C6786, Burkholderia oklahomensis E0147, Burkholderiaphymatum STM815, Burkholderia pseudomallei 1106a, Burkholderiapseudomallei 1106b, Burkholderia pseudomallei 14, Burkholderiapseudomallei 1655, Burkholderia pseudomallei 1710b, Burkholderiapseudomallei 305, Burkholderia pseudomallei 406e, Burkholderiapseudomallei 668, Burkholderia pseudomallei 7894, Burkholderiapseudomallei K96243, Burkholderia pseudomallei NCTC 13177, Burkholderiasp. 383, Burkholderia thailandensis Bt4, Burkholderia thailandensisE264, Burkholderia thailandensis MSMB43, Burkholderia thailandensisTXDOH, Burkholderia ubonensis Bu, Burkholderia vietnamiensis G4,Caulobacter crescentus CB15, Cellvibrio japonicus Ueda107,Chromobacterium violaceum ATCC 12472, Chromohalobacter salexigens DSM3043, Citrobacter koseri ATCC BAA-895, Comamonas testosteroni KF-1,Cupriavidus taiwanensis, Dechloromonas aromatica RCB, Delftiaacidovorans SPH-1, Desulfovibrio desulfuricans subsp. desulfuricans str.G20, Desulfovibrio desulfuricans subsp. desulfuricans str. G20,Enterobacter cancerogenus ATCC 35316, Enterobacter sakazakii ATCCBAA-894, Enterobacter sp. 638, Erwinia tasmaniensis, Escherichiaalbertii TWO7627, Escherichia coli O157:H7 EDL933, Escherichia coliO157:H7 str.EC4024, Escherichia coli O157:H7 str. EC4196, gammaproteobacterium HTCC5015, gamma proteobacterium KT 71, Geobacter sp.M21, Hahella chejuensis KCTC 2396, Halorhodospira halophila SL1,Herminiimonas arsenicoxydans, Idiomarina baltica OS145, Idiomarinaloihiensis L2TR, Janthinobacterium sp. Marseille, Klebsiella pneumoniae342, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Klebsiella sp.ZD414, Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130,Leptospira interrogans serovar Lai str. 56601, Leptothrix cholodniiSP-6, Limnobacter sp. MED105, Magnetospirillum magneticum AMB-1, marinegamma proteobacterium HTCC2080, marine gamma proteobacterium HTCC2143,marine gamma proteobacterium HTCC2207, marine metagenome, Marinobacteralgicola DG893, Marinobacter aquaeolei VT8, Marinobacter sp. ELB17,Marinomonas sp. MWYL1, Methylibium petroleiphilum PM1, Methylobacillusflagellatus KT, Methylobacterium chloromethanicum CM4, Methylobacteriumextorquens PA1, Methylobacterium populi BJ001, Methylocella silvestrisBL2, Methylococcus capsulatus str. Bath, Moritella sp. PE36, Nitrobactersp. Nb-311A, Nitrobacter winogradskyi Nb-255, Nitrococcus mobilisNb-231, Nitrosococcus oceani ATCC 19707, Nitrosococcus oceani C-27,Nitrosomonas europaea ATCC 19718, Nitrosomonas eutropha C91,Nitrosospira multiformis ATCC 25196, Oceanospirillum sp. MED92,Oligotropha carboxidovorans OM5, Pectobacterium atrosepticum SCRI1043,Photobacterium profundum 3TCK, Photobacterium profundum SS9,Photobacterium sp. SKA34, Photorhabdus luminescens, Photorhabdusluminescens subsp. laumondii TTO1, Polaromonas naphthalenivorans CJ2,Polaromonas sp. JS666, Polynucleobacter sp. QLW-P1DMWA-1, Proteusmirabilis HI4320, Providencia stuartii ATCC 25827, Pseudoalteromonasatlantica T6c, Pseudoalteromonas haloplanktis TAC125, Pseudoalteromonassp. 643A, Pseudoalteromonas tunicata D2, Pseudomonas aeruginosa PA7,Pseudomonas aeruginosa PACS2, Pseudomonas aeruginosa PA01, Pseudomonasaeruginosa UCBPP-PA14, Pseudomonas entomophila L48, Pseudomonasfluorescens Pf0-1, Pseudomonas fluorescens Pf-5, Pseudomonas mendocinaymp, Pseudomonas putida F1, Pseudomonas putida GB-1, Pseudomonas putidaKT2440, Pseudomonas putida W619, Pseudomonas stutzeri A1501, Pseudomonassyringae pv. Phaseolicola 1448A, Pseudomonas syringae pv. syringaeB728a, Pseudomonas syringae pv. tomato str. DC3000, Psychromonasingrahamii 37, Ralstonia eutropha H16, Ralstonia eutropha JMP134,Ralstonia metallidurans CH34, Ralstonia pickettii 12D, Ralstoniapickettii 12J, Ralstonia solanacearum GMI1000, Ralstonia solanacearumIP01609, Ralstonia solanacearum MolK2, Ralstonia solanacearum UW551,Reinekea sp. MED297, Rhodobacterales bacterium Y4I, Rhodoferaxferrireducens T118, Rhodopseudomonas palustris BisA53, Rhodopseudomonaspalustris BisB18, Rhodopseudomonas palustris BisB5, Rhodopseudomonaspalustris CGA009, Rhodopseudomonas palustris HaA2, Rhodopseudomonaspalustris TIE-1, Rhodospirillum centenum SW, Saccharophagus degradans2-40, Salmonella enterica subsp. arizonae serovar 62:z4,z23:—,Salmonella enterica subsp. enterica serovar Choleraesuis str. SC-B67,Salmonella enterica subsp. enterica serovar allinarum str. 287/91,Salmonella enterica subsp. enterica serovar Hadar str. RI 05P066,Salmonella enterica subsp. enterica serovar Javiana str. GA_MM04042433,Salmonella enterica subsp. enterica serovar Saintpaul str. SARA23,Salmonella enterica subsp. enterica serovar Saintpaul str. SARA29,Salmonella enterica subsp. enterica serovar Typhi str. CT18, Salmonellatyphimurium LT2, Serratia proteamaculans 568, Shewanella amazonensisSB2B, Shewanella baltica OS155, Shewanella baltica OS185, Shewanellabaltica OS195, Shewanella baltica 0S223, Shewanella benthica KT99,Shewanella denitrificans OS217, Shewanella frigidimarina NCIMB 400,Shewanella halifaxensis HAW-EB4, Shewanella loihica PV-4, Shewanellaoneidensis MR-1, Shewanella pealeana ATCC 700345, Shewanellaputrefaciens 200, Shewanella sediminis HAW-EB3, Shewanella sp. ANA-3,Shewanella sp. MR-4, Shewanella sp. MR-7, Shewanella sp. W3-18-1,Shewanella woodyi ATCC 51908, Shigella boydii Sb227, Shigelladysenteriae Sd197, Stenotrophomonas maltophilia K279a, Stenotrophomonasmaltophilia R551-3, Streptococcus sp. (N1), synthetic construct, Thauerasp. MZ1T, Thioalkalivibrio sp. HL-EbGR7, Thiobacillus denitrificansATCC25259, Thiomicrospira crunogena XCL-2, Vibrio alginolyticus 12G01,Vibrio angustum S14, Vibrio campbellii AND4, Vibrio cholerae 2740-80,Vibrio cholerae MZO-2, Vibrio cholerae 01 biovar eltor str. N16961,Vibrio cholerae V51, Vibrio fischeri ES114, Vibrio fischeri MJ11, Vibrioharveyi ATCC BAA-1116, Vibrio mimicus, Vibrionales bacterium SWAT-3,Vibrio parahaemolyticus AQ3810, Vibrio parahaemolyticus RIMD 2210633,Vibrio shilonii AK1, Vibrio splendidus 12B01, Vibrio sp. MED222, Vibriovulnificus CMCP6, Vibrio vulnificus YJ016, Xanthomonas axonopodis pv.citri str. 306, Xanthomonas campestris pv. campestris str.ATCC 33913,Xanthomonas campestris pv. campestris str. B100, Xanthomonas campestrispv. Vesicatoria str. 85-10, Xanthomonas oryzae pv. oryzae KACC10331,Xanthomonas oryzae pv. oryzae PXO99A, Xanthomonas oryzae pv. oryzicolaBLS256, Yersinia bercovieri ATCC 43970, Yersinia enterocolitica subsp.enterocolitica 8081, Yersinia frederiksenii ATCC 33641, Yersiniaintermedia ATCC 29909, Yersinia mollaretii ATCC 43969, Yersinia pestisAngola, Yersinia pestis biovar Orientalis str. F1991016, Yersinia pestisC092, Yersinia pestis KIM or Yersinia pseudotuberculosis IP 31758.

In one embodiment of the invention, the precursor thioesterase is athioesterase that has an analogous sequence to that of 'TesA (e.g., aTesA enzyme sans the signal peptide). In a preferred embodiment, theprecursor thioesterase has at least about 20%, for example, at leastabout 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to'TesA. In yet another example, the precursor thioesterase has at leastabout 20%, for example, at least about 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to a 'TesA that is obtained from anE. coli, such as an E. coli K12. In a further example, the precursorthioesterase is a thioesterase that has an analogous sequence to thesequence of SEQ ID NO:31 in FIG. 58, and preferably at least about 20%,for example, at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:31 in FIG. 58. The analogous sequence canbe from a naturally-occurring protein or can be from a previouslymodified protein.

In one embodiment of the invention, the precursor thioesterase is athioesterase that comprises the amino acid strings:

G-D-S-L-X(5)-M (SEQ ID NO:28), wherein:

the “X” refers to any amino acid residue; the number in theparenthetical adjacent thereto,

when present, refers to the number of X residues in the stretch of aminoacid residues;

the S residue at position 3 is a catalytic residue;

the D residue at position 2 may be substituted with N or T;

the L residue at position 4 may be substituted with C or Q;

the M residue at position 10 may be substituted with C, D, L, N, T, orV;

and/orV-X(2) G X N D XL (SEQ ID NO:29), wherein:

each “X” refers to any amino acid residue; the number in the parenthesesadjacent thereto, when present, refers to the number of X residues inthe stretch of amino acid residues;

the N residue at position 6 is in the oxyanion hole;

the V residue at position 1 may be substituted with L;

the N residue at position 6 may be substituted with V, L, C, A, G, H, I,T, or W;

the D residue at position 7 may be substituted with E;

the L residue at position 9 may be substituted with I, W, F, T, M, A, E,N, or V;

and/orD-X(2)-H-P-X(7)-I (SEQ ID NO:30), wherein:

each “X” refers to any amino acid residue; each number in theparentheses adjacent thereto, when present, refers to the number of Xresidues in the respective stretch of amino acid residues;

the D and H residues at positions 1 and 4 respectively are the catalyticresidues;

the P residue at position 5 may be substituted with G, A, F, L, S, or V;

the I residue at position 13 may be substituted with L or V.

In one embodiment of the invention, the precursor thioesterase is athioesterase having immunological cross-reactivity with a 'TesA obtainedfrom an E. coli. In a particular embodiment, the precursor thioesterasehas immunological cross-reactivity with the 'TesA obtained from an E.coli K-12. In a particular embodiment, the precursor thioesterase hasimmunological cross-reactivity with a thioesterase comprising the aminoacid sequence of SEQ ID NO:31 as set forth in FIG. 58. In a particularembodiment, the precursor thioesterase has cross-reactivity withfragments (or portions) of any of the thioesterases obtained from an E.coli, or from an E. coli K-12, and/or of any thioesterase that comprisesthe amino acid sequence of SEQ ID NO:31 as set forth in FIG. 58. Theprecursor enzyme having immunological cross-reactivity with 'TesA can bea naturally-occurring protein, a previously modified protein, or asynthetic protein.

In another particular example, the precursor thioesterase is a 'TesAfrom an E. coli, or is a homolog, a paralog or an ortholog of a 'TesAfrom an E. coli, such as a 'TesA from an E. coli K12. The thioesteraseprecursor from which a mutant of the present invention is derived canalso be an enzymatically active portion or a fragment of any one of theafore-described thioesterases.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided that comprises anamino acid sequence having at least one substitution of an amino acid,as compared to a precursor thioesterase, such that the mutantthioesterase has at least one altered property in relation to theprecursor thioesterase. In an exemplary embodiment of the invention, amutant thioesterase is provided that has an amino acid sequence with asingle substitution mutation, and exhibits at least one altered propertyas compared to the precursor thioesterase from which the mutant isderived. In an exemplary embodiment of the invention, a mutantthioesterase is provided that comprises an amino acid sequence havingtwo or more substitution mutations from the sequence of its precursorthioesterase, and the mutant thioesterase has at least one alteredproperty as compared to the precursor thioesterase.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is a variantof a precursor thioesterase, and which has at least one altered propertyin vitro or in vivo in relation to such a precursor thioesterase,wherein the precursor thioesterase is a thioesterase that comprises ananalogous sequence to SEQ ID NO:31 in FIG. 58 and accordingly comprisescorresponding amino acid residues 1-182 of SEQ ID NO:31, and wherein theprecursor thioesterase is modified at one or more amino acid positionsselected from positions corresponding to one or more residues 1-182 ofSEQ ID NO:31 in FIG. 58.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is a variantof a precursor thioesterase that comprises an analogous sequence to SEQID NO:31 in FIG. 58 and accordingly comprises corresponding amino acidresidues 1-182 of SEQ ID NO:31, and which has at least one alteredproperty in vitro or in vivo in relation to such precursor thioesterase,wherein the precursor thioesterase is mutated at one or more positionscorresponding to one or more amino acid positions of SEQ ID NO:31 inFIG. 58 selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, and/or 182.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent) is provided, which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 of FIG. 58 and accordingly comprises corresponding amino acidresidues 1-182 of SEQ ID NO:31, and which has at least one alteredproperty in vitro or in vivo in relation to such precursor thioesterase,wherein the precursor thioesterase is mutated with one or moresubstitutions selected from A1C, A1F, A1L, A1Q, A1R, A1S, A1V, A1Y, D2E,D2H, D2K, D2L, D2M, D2P, D2R, D2W, T3E, T3G, T3K, T3L, T3R, T3W, L4A,L4G, L4M, L4N, L4S, L4V, L4Y, L5C, L5E, L5F, L5G, L5H, L5K, L5N, L5Q,L5S, L5W, L5Y, I6A, I6L, I6T, I6V, L7A, L7C, L7E, L7K, L7M, L7N, L7S,L7T, L7V, L7W, L7Y, G8A, G8K, G8S, D9N, D9T, S10C, L11A, L11C, L11I,L11M, L11Q, Lily, S12A, S12I, S12L, S12M, S12N, S12T, S12V, S12Y, A13C,A13D, A13G, A13H, A13I, A13L, A13N, A13S, A13T, A13V, A13W, A13Y, G14A,G14C, G14E, G14F, G14I, G14K, G14M, G14N, G14P, G14Q, G14R, G14S, G14T,G14V, Y15A, Y15C, Y15D, Y15E, Y15G, Y15I, Y15L, Y15M, Y15N, Y15Q, Y15R,Y15S, Y15V, R16A, R16D, R16E, R16G, R16H, R16I, R16L, R16M, R16N, R16P,R16Q, R16S, R16T, R16V, R16W, M17A, M17C, M17D, M17E, M17G, M17K, M17L,M17N, M17P, M17Q, M17R, M17S, M17T, M17V, S18E, S18M, S18N, S18T, A19C,A19E, A19L, A19V, S20A, S20C, S20D, S20G, S20L, S20T, S20W, A21G, A21I,A21L, A21P, A21Y, A22C, A22D, A22E, A22F, A22G, A22H, A22I, A22K, A22L,A22M, A22N, A22P, A22R, A22S, A22T, A22Y, W23A, W23H, W23N, W23P, W23Y,P24A, P24C, P24D, P24E, P24F, P24G, P24I, P24M, P24N, P24S, P24T, P24V,P24W, A25D, A25E, A25L, A25N, A25P, A25Q, A25R, A25S, A25V, L26C, L26D,L26E, L26F, L26G, L26H, L26I, L26K, L26N, L26P, L26Q, L26R, L26S, L26V,L26W, L26Y, L27A, L27C, L27F, L27H, L27M, L27R, L27S, L27T, L27V, L27W,L27Y, N28A, N28G, N28I, N28K, N28M, N28P, N28R, N28W, D29M, D29P, D29V,K30P, W31D, W31E, W31G, W31L, W31N, W31P, W31R, W31S, W31T, Q32V, Q32Y,S33F, S33G, S33I, S33M, S33R, K34A, K34H, K34M, K34R, T35F, T35G, T35K,T35L, T35M, T35Q, T35V, T35Y, S36A, S36F, S36H, S36I, S36L, S36W, V37A,V37F, V37G, V37H, V37L, V37N, V37S, V37Q, V37S, V37W, V37Y, V38D, V38E,V38F, V38G, V38K, V38L, V38P, V38R, V38S, N39A, N39C, N39E, N39F, N39G,N39K, N39M, N39P, N39Q, N39R, N39T, N39V, N39W, N39Y, A40D, A40G, A40H,A40L, A40M, A40P, A40T, A40V, A40Y, S41C, S41P, S41T, I42A, I42C, I42D,I42E, I42G, I42K, I42L, I42M, I42P, I42S, I42T, I42W, I42Y, S43A, S43C,S43D, S43E, S43F, S43G, S43H, S43L, S43M, S43N, S43P, S43R, S43T, S43V,S43W, G44A, G44C, G44E, G44F, G44H, G44K, G44L, G44M, G44N, G44Q, G44R,G44S, G44W, G44Y, D45A, D45C, D45E, D45F, D45G, D45H, D45I, D45K, D45L,D45M, D45P, D45Q, D45S, D45T, D45V, D45W, T46A, T46C, T46D, T46E, T46F,T46G, T46I, T46K, T46L, T46N, T46R, T46S, T46V, T46W, S47A, S47C, S47E,S47F, S47G, S47L, S47M, S47P, S47Q, S47R, S47T, S47V, S47W, S47Y, Q48C,Q48D, Q48E, Q48F, Q48G, Q48I, Q48M, Q48S, Q48T, Q48V, Q48W, Q48Y, Q49A,Q49C, Q49D, Q49E, Q49G, Q49H, Q49I, Q49K, Q49L, Q49M, Q49P, Q49R, Q49S,Q49V, Q49W, Q49Y, G50A, G50C, G50E, G50F, G50I, G50K, G50L, G50M, G50N,G50P, G50Q, G50R, G50S, G50T, G50W, G50Y, L51A, L51C, L51D, L51F, L51H,L51N, L51P, L51S, L51T, L51V, L51W, L51Y, A52C, A52D, A52H, A52I, A52L,A52M, A52P, A52R, A52V, A52W, A52Y, R53A, R53C, R53D, R53E, R53F, R53G,R53I, R53K, R53L, R53N, R53S, R53T, R53V, R53W, R53Y, L54A, L54C, L54E,L54F, L54G, L54M, L54N, L54S, L54T, L54W, L54Y, P55A, P55G, P55Y, A56P,A56R, A56W, A56Y, L57A, L57C, L57F, L57G, L57H, L57I, L57K, L57N, L57P,L57Q, L57R, L57S, L57T, L57V, L57W, L57Y, L58A, L58D, L58E, L58F, L58G,L58H, L58I, L58M, L58N, L58R, L58S, L58V, L58W, L58Y, K59E, K59R, K59V,Q60E, Q60M, Q60P, H61A, H61D, H61E, H61G, H61P, H61W, Q62G, Q62M, Q62P,Q62W, P63D, P63E, P63G, P63I, P63K, P63L, P63M, P63N, P63Q, P63R, P63S,P63T, P63V, P63W, R64D, R64E, R64F, R64L, R64M, R64P, R64Q, R64W, R64Y,W65A, W65E, W65G, W65K, W65L, W65M, W65N, W65P, W65R, W65V, V66C, V66G,V66I, V66M, V66N, V66Q,V66S, V66W, V66Y, L67A, L67C, L67E, L67G, L67M,L67Q, L67Q, L67S, L67T, L67W, V68A, V68E, V68G, V68L, V68M, V68N, V68P,V68Q, V68S, V68T, E69A, E69C, E69D, E69F, E69G, E69H, E69K, E69L, E69M,E69N, E69P, E69Q, E69S, E69V, E69W, E69Y, L70A, L70C, L70E, L70F, L70G,L70H, L70I, L70K, L70Q, L70S, L70T, L70V, L70W, G71A, G71C, G71S, G72A,G72C, G72M, G72P, G72S, N73A, N73C, N73G, N73H, N73I, N73L, N73P, N73R,N73S, N73T, N73V, N73W, D74A, D74C, D74E, D74F, D74G, D74Q, D74S, D74W,D74Y, G75A, G75C, G75D, G75E, G75F, G75I, G75K, G75L, G75M, G75N, G75P,G75R, G75T, G75V, G75W, G75Y, L76A, L76C, L76D, L76E, L76F, L76G, L76I,L76K, L76M, L76N, L76P, L76Q, L76R, L76T, L76V, L76W, R77A, R77C, R77D,R77E, R77F, R77G, R77H, R77K, R77L, R77N, R77Q, R77S, R77V, R77W, G78A,G78C, G78D, G78E, G78F, G78M, G78N, G78P, G78Q, G78R, G78S, G78T, G78V,G78Y, F79A, F79D, F79E, F79G, F79H, F79K, F79M, F79N, F79P, F79Q, F79S,F79V, F79W, F79Y, Q80A, Q80E, Q80G, Q80L, Q80M, Q80S, Q80W, Q80Y, P81A,P81E, P81K, P81L, P81M, P81N, P81T, P81W, P81Y, Q82A, Q82F, Q82I, Q82M,Q82N, Q82P, Q82R, Q82S, Q82T, Q82V, Q82W, Q82Y, Q83A, Q83C, Q83F, Q83G,Q83K, Q83L, Q83M, Q83N, Q83R, Q83S, Q83T, Q83V, Q83W, Q83Y, T84A, T84D,T84E, T84F, T84G, T84H, T84K, T84L, T84M, T84N, T84Q, T84R, T84S, T84V,T84W, T84Y, E85A, E85C, E85D, E85F, E85G, E85L, E85P, E85Q, E85R, E85S,E85T, E85V, E85W, E85Y, Q86A, Q86G, Q86H, Q86K, Q86P, Q86T, Q86V, Q86W,Q86Y, T87A, T87C, T87D, T87E, T87F, T87G, T87H, T87L, T87M, T87P, T87R,T87S, T87V, T87W, L88A, L88C, L88E, L88F, L88G, L88H, L88Q, L88S, L88W,L88Y, R89A, R89G, R89H, R89L, R89P, R89T, R89V, R89W, Q90E, Q90L, Q90N,Q90P, Q90W, Q90Y, 191E, I91G, I91L, I91M, I91N, I91Q, I91S, I91V, I91Y,L92A, L92C, L92E, L92G, L92H, L92N, L92Q, L92R, L92S, L92T, L92V, L92Y,Q93A, Q93E, Q93F, Q93G, Q93H, Q93I, Q93L, Q93M, Q93N, Q93P, Q93S, Q93V,Q93W, Q93Y, D94C, D94E, D94F, D94G, D94H, D94K, D94L, D94N, D94P, D94Q,D94R, D94S, D94V, V95A, V95C, V95D, V95E, V95F, V95G, V95I, V95L, V95M,V95N, V95P, V95Q, V95T, V95W, V95Y, K96A, K96C, K96L, K96N, K96P, K96Q,K96R, K96V, K96Y, A97C, A97E, A97F, A97K, A97N, A97P, A97R, A97V, A97W,A98E, A98G, A98K, A98L, A98P, A98V, A98W, A98Y, N99A, N99C, N99D, N99G,N99L, N99M, N99P, N99Q, N99R, N99S, N99W, N99Y, A100D, A100E, A100G,A100H, A100I, A100K, A100L, A100M, A100Q, A100R, A100S, A100T, A100V,A100W, A100Y, E101A, E101D, E101G, E101L, E101M, E101P, E101S, E101T,E101V, P102E, P102F, P102G, P102H, P102I, P102L, P102Q, P102R, P102S,P102V, P102W, P102Y, L103A, L103C, L103E, L103G, L103I, L103K, L103N,L103Q, L103R, L103S, L103T, L103V, L103W, L104A, L104C, L104E, L104G,L104I, L104N, L104P, L104Q, L104S, L104W, L104Y, M105A, M105C, M105E,M105F, M105G, M105I, M105K, M105L, M105P, M105T, M105V, M105W, Q106A,Q106C, Q106D, Q106G, Q106H, Q106K, Q106L, Q106M, Q106R, Q106S, Q106T,Q106V, Q106W, Q106Y, I107A, I107C, I107E, I107F, I107G, I107K, I107L,I107M, I107Q, I107S, I107T, I107V, I107Y, R108A, R108C, R108D, R108E,R108F, R108G, R108H, R108I, R108L, R108M, R108S, R108V, R108W, R108Y,L109A, L109C, L109D, L109E, L109F, L109G, L109K, L109M, L109P, L109Q,L109R, L109S, L109T, L109V, L109Y, P110A, P110C, P110D, P110E, P110F,P110G, P110H, P110K, P110L, P110M, P110N, P110R, P110S, P110V, P110W,A111C, A111E, A111L, A111M, A111N, A111P, A111Q, A111R, A111S, A111V,A111W, A111Y, N112A, N112F, N112G, N112I, N112K, N112L, N112P, N112R,N112V, N112W, N112Y, Y113A, Y113C, Y113D, Y113E, Y113G, Y113I, Y113M,Y113P, Y113Q, Y113S, Y113S, Y113W, G114A, G114F, G114K, G114L, G114M,G114P, G114W, G114Y, R115A, R115C, R115E, R115G, R115I, R115N, R115P,R115Q, R115S, R115V, R115W, R115Y, R116C, R116D, R116E, R116H, R116T,R116V, R116W, Y117A, Y117C, Y117D, Y117E, Y117G, Y117H, Y117I, Y117L,Y117M, Y117N, Y117P, Y117Q, Y117R, Y117S, Y117T, Y117V, Y117W, N118A,N118C, N118E, N118F, N118G, N118H, N118I, N118K, N118L, N118M, N118P,N118Q, N118S, N118T, N118V, N118W, E119C, E119D, E119F, E119G, E119K,E119L, E119M, E119P, E119Q, E119R, E119T, E119W, E119Y, A120D, A120E,A120G, A120I, A120L, A120P, A120T, A120W, F121A, F121C, F121D, F121E,F121G, F121K, F121L, F121M, F121N, F121P, F121Q, F121R, F121S, F121V,F121W, F121Y, S122A, S122C, S122D, S122E, S122F, S122G, S122I, S122L,S122M, S122P, S122R, S122V, S122W, S122Y, A123C, A123E, A123F, A123H,A123L, A123R, A123T, A123V, A123W, A123Y, I124A, I124C, I124D, I124E,I124G, I124H, I124K, I124L, I124R, I124S, I124T, I124W, I124Y, Y125C,Y125F, Y125G, Y125H, Y125I, Y125L, Y125P, Y125Q, Y125R, Y125S, Y125T,Y125V, Y125W, P126C, P126F, P126H, P126K, P126R, P126T, P126V, P126Y,K127A, K127I, K127P, K127S, L128A, L128C, L128E, L128F, L128G, L128Q,L128R, L128S, L128T, L128V, L128W, A129D, A129F, A129H, A129I, A129K,A129L, A129N, A129W, A129Y, K130E, K130I, K130P, K130V, E131A, E131C,E131D, E131F, E131G, E131I, E131K, E131L, E131N, E131P, E131V, E131W,F132C, F132D, F132E, F132K, F132L, F132N, F132P, F132T, F132V, D133C,D133K, D133R, D133S, D133T, D133V, D133Y, V134C, V134D, V134E, V134I,V134K, V134M, V134N, V134P, V134Q, V134R, V134S, V134W, V134Y, P135A,P135E, P135K, P135Q, L136A, L136C, L136D, L136E, L136F, L136G, L136H,L136K, L136M, L136N, L136P, L136Q, L136R, L136S, L136T, L137A, L137C,L137D, L137E, L137G, L137H, L137K, L137P, L137Q, L137R, L137S, L137Y,P138E, P138F, P138G, P138N, P138R, P138T, P138V, F139A, F139C, F139D,F139E, F139G, F139H, F139L, F139M, F139N, F139S, F139T, F139V, F139W,F140A, F140C, F140G, F140I, F140L, F140M, F140N, F140P, F140S, F140T,F140V, F140W, M141A, M141C, M141D, M141E, M141F, M141G, M141K, M141L,M141P, M141Q, M141R, M141T, M141V, M141W, M141Y, E142A, E142C, E142G,E142I, E142L, E142M, E142N, E142P, E142Q, E142R, E142S, E142T, E142V,E142W, E142Y, E143A, E143D, E143F, E143G, E143I, E143M, E143P, E143W,V144A, V144D, V144E, V144G, V144H, V144N, V144P, V144Q, V144R, V144S,V144W, V144Y, Y145A, Y145C, Y145D, Y145E, Y145G, Y145I, Y145L, Y145M,Y145N, Y145Q, Y145R, Y145S, Y145T, Y145W, L146A, L146C, L146D, L146E,L146G, L146H, L146P, L146S, L146W, K147G, K147P, K147R, K147W, P148D,P148E, P148W, Q149L, W150C, W150D, W150E, W150G, W150L, W150P, W150Q,W150R, W150T, M150V, M151A, M151C, M151D, M151E, M151F, M151G, M151I,M151L, M151Q, M151R, M151S, M151T, M151V, M151W, Q152A, Q152D, Q152E,Q152F, Q152H, Q152I, Q152K, Q152L, Q152N, Q152P, Q152R, Q152S, Q152T,Q152V, Q152Y, D153A, D153E, D153F, D153I, D153K, D153M, D153P, D153Q,D153V, D153W, D154A, D154C, D154E, D154F, D154G, D154H, D154I, D154K,D154L, D154M, D154N, D154P, D154R, D154S, D154T, D154V, D154W, G155A,G155F, G155H, G155I, G155P, G155V, G155W, G155Y, I156A, I156C, I156E,I156F, I156G, I156K, I156L, I156M, I156Q, I156R, I156S, I156T, I156V,I156Y, H157C, H157E, P158A, P158F, P158G, P158H, P158I, P158L, P158Q,P158S, P158T, P158V, P158W, N159C, N159E, N159G, N159I, N159K, N159L,N159M, N159P, N159Q, N159R, N159T, N159V, N159W, R160A, R160C, R160D,R160E, R160G, R160H, R160I, R160K, R160N, R160Q, R160S, R160W, D161E,D161G, D161I, D161K, D161L, D161M, D161N, D161Q, D161R, D161S, D161V,D161W, A162G, A162I, A162K, A162L, A162N, A162R, A162T, A162V, A162Y,Q163A, Q163C, Q163D, Q163E, Q163F, Q163G, Q163I, Q163L, Q163M, Q163S,Q163T, Q163V, Q163W, Q163Y, P164A, P164C, P164D, P164K, P164L, P164M,P164N, P164R, P164T, P164V, P164W, F165D, F165E, F165G, F165H, F165I,F165K, F165L, F165M, F165R, F165S, F165T, F165V, F165W, F165Y, I166A,I166C, I166F, I166L, I166M, I166S, I166V, I166Y, A167C, A167D, A167E,A167F, A167G, A167K, A167L, A167M, A167N, A167Q, A167R, A167T, A167V,A167W, A167Y, D168A, D168G, D168H, D168L, D168M, D168P, D168R, D168T,D168V, D168W, W169A, W169D, W169E, W169G, W169K, W169M, W169Q, W169R,W169S, W169T, W169V, M170A, M170E, M170F, M170G, M170H, M170L, M170N,M170Q, M170S, M170T, M170V, M170W, M170Y, A171E, A171F, A171I, A171S,A171V, A171W, K172A, K172M, K172P, Q173D, Q173I, Q173N, Q173P, Q173W,Q173Y, L174A, L174F, L174G, L174Q, L174S, L174T, L174W, L174W, L174Y,Q175F, Q175I, Q175L, Q175M, Q175Y, P176D, P176H, P176K, P176L, P176N,P176Q, P176R, P176V, P176W, P176Y, L177D, L177F, L177G, L177M, L177S,L177T, V178A, V178F, V178G, V178K, V178L, V178R, V178S, V178T, V178W,N179G, N179H, N179R, N179T, N179V, N179W, N179Y, H180A, H180E, H180G,H180L, H180P, H180R, H180S, H180V, H180W, D181A, D181C, D181E, D181G,D181H, D181I, D181L, D181P, D181Q, D181R, D181S, D181T, D181W, S182A,S182C, S182D, S182E, S182G, S182I, S182K, S182L, S182N, S182P, S182Q,S182R, S182T, and/or S180V, wherein the numbers in the substitutionmutation designations refer to amino acid positions of SEQ ID NO:31.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₀ substrates (i.e., substrates, the carbonchains of which are 10 carbons in length), and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding to one or more of residuesselected from 5-30, 35-60, 65-98, 102-139, and/or 140-180 of SEQ IDNO:31. The increased substrate specificity for, and/or activity withrespect to C₁₀ substrates can be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₀ substrates, and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding to one or more residues ofSEQ ID NO:31 selected from 1, 3, 4, 7, 9, 12, 13, 14, 16, 17, 20, 22,24, 25, 28, 32, 38, 39, 40, 42, 43, 46, 47, 48, 49, 50, 51, 52, 54, 56,59, 60, 64, 68, 72, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 138, 139,140, 141, 142, 144, 145, 146, 147, 148, 150, 151, 152, 156, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,175, 176, 177, 178, 179, 180, 181, and/or 182. The increased substratespecificity for, and/or activity with respect to C₁₀ substrates can bemeasured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent) is provided, which has an increasedsubstrate specificity for, and/or activity (e.g., catalytic rate) withrespect to C₁₀ substrates, and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated with one or moresubstitutions selected from A1L, A1S, T3K, L4A, L7M, L7V, D9N, S12A,A13D, G14A, G14E, G14P, G14Q, G14R, G14S, G14V, R16G, R16L, R16M, R16N,R16P, R16Q, R16T, M17C, M17L, M17T, M17V, S20A, S20C, S20D, S20G, S20L,S20T, S20W, A22C, A22D, A22E, A22G, A22H, A22I, A22K, A22N, P24A, P24C,P24D, P24F, P24I, P24S, P24T, P24V, P24W, A25E, A25L, A25N, A25Q, A25V,N28A, N28R, Q32V, Q32Y, V38E, V38K, V38R, N39A, N39T, A40D, A40H, I42A,I42E, I42L, I42S, I42T, I42W, I42Y, S43A, S43C, S43D, S43E, S43L, S43N,S43P, T46E, T46F, T46I, T46L, T46V, S47A, S47C, S47F, S47G, S47L, S47M,S47T, S47V, Q48D, Q48E, Q48G, Q48S, Q48T, Q48V, Q48W, Q49A, Q49C, Q49D,Q49G, Q49H, Q49L, Q49M, Q49S, G50A, G50Q, L51A, L51F, L51H, L51Y, A52D,A52M, L54T, A56P, K59R, Q60M, R64D, R64E, R64Q, V68L, G72A, G72C, G72P,G72S, G75A, G75C, G75D, G75E, G75F, G75I, G75K, G75L, G75M, G75N, G75P,G75T, G75V, G75W, G75Y, L76A, L76D, L76G, L76I, L76K, L76M, L76N, L76P,L76Q, L76R, L76W, R77G, R77L, R77Q, G78A, G78C, G78E, G78F, G78M, G78N,G78Q, G78R, G78S, G78T, G78V, G78Y, F79A, F79D, F79E, F79G, F79H, F79N,F79Q, F79W, F79Y, Q80E, P81N, P81T, P81Y, Q82R, Q82S, Q82T, Q83A, Q83C,Q83F, Q83G, Q83K, Q83L, Q83M, Q83N, Q83R, Q83S, Q83T, Q83V, Q83W, Q83Y,T84A, T84F, T84L, T84M, T84N, T84Q, T84V, T84Y, E85A, E85C, E85L, E85Q,E85R, E85S, E85T, E85W, E85Y, Q86A, Q86G, Q86K, Q86T, T87D, T87P, R89A,R89G, Q90E, Q90Y, I91V, L92V, Q93A, Q93E, Q93G, Q93H, Q93I, Q93L, Q93S,Q93W, Q93Y, D94E, D94F, D94G, D94H, D94K, D94N, D94Q, D94R, D94S, D94V,V95L, V95T, K96V, K96Y, A98W, N99G, N99L, N99P, N99Q, N99R, N99Y, A100G,A100V, E101A, E101D, E101G, E101L, E101M, E101S, E101T, E101V, P102S,L103G, M105C, M105I, M105V, Q106A, Q106D, Q106H, Q106W, I107Y, R108A,R108D, R108E, R108F, R108G, R108H, R108I, R108L, R108M, R108S, R108W,R108Y, L109A, L109D, L109E, L109F, L109G, L109K, L109P, L109R, L109S,L109Y, P110C, P110D, P110E, P110F, P110G, P110H, P110K, P110L, P110M,P110N, P110R, P110S, P110V, P110W, A111C, A111E, A111L, A111M, A111P,A111Q, A111R, A111V, A111W, A111Y, N112A, N112F, N112G, N112K, N112R,N112W, Y113A, Y113C, Y113G, Y113I, Y113M, G114K, G114L, G114P, R115A,R115C, R115E, R115G, R115N, R115S, R115W, R115Y, R116D, R116E, R116W,Y117A, Y117C, Y117E, Y117I, Y117L, Y117N, Y117Q, Y117R, Y117S, Y117T,Y117V, N118C, N118G, N118I, N118K, N118S, N118T, N118V, N118W, E119C,E119F, E119G, E119K, E119M, E119R, E119W, E119Y, A120D, A120E, A120G,A120W, F121A, F121D, F121E, F121M, F121P, F121Q, F121R, F121S, F121Y,S122D, S122E, S122F, S122I, S122L, S122M, S122V, S122W, S122Y, A123H,A123L, A123V, I124T, Y125C, Y125F, Y125G, Y125P, Y125S, Y125V, Y125W,P126R, P126T, P126V, P126Y, K127S, L128C, L128T, L128V, K130E, K130I,K130V, F132D, F132E, F132N, F132T, D133K, D133R, D133S, D133T, D133V,D133Y, V134I, V134M, V134S, P138E, P138N, P138R, P138T, P138V, F139A,F139D, F139G, F139H, F139M, F139S, F139W, F140C, F140G, F140M, F140N,F140P, F140S, M141A, M141C, M141D, M141E, M141F, M141G, M141K, M141L,M141P, M141Q, M141R, M141T, M141V, M141W, M141Y, E142A, E142C, E142P,E142Q, E142W, E142Y, V144D, V144E, V144G, V144H, V144N, V144P, V144Q,V144R, V144S, V144W, V144Y, Y145A, Y145C, Y145D, Y145E, Y145G, Y145I,Y145L, Y145M, Y145N, Y145Q, Y145T, Y145W, L146A, L146C, L146D, L146E,L146G, L146H, L146S, L146W, K147G, K147P, K147W, P148D, P148E, W150C,W150D, W150E, W150G, W150L, W150Q, W150T, M151A, M151C, M151E, M151F,M151G, M151I, M151Q, M151S, M151T, M151V, M151W, Q152D, Q152F, Q152I,Q152L, Q152T, I156L, P158A, P158F, P158G, P158H, P158I, P158L, P158Q,P158T, P158V, N159C, N159E, N159G, N159I, N159K, N159L, N159M, N159R,N159T, N159V, R160A, R160C, R160D, R160E, R160G, R160H, R160N, R160Q,R160S, R160W, D161E, D161G, D161I, D161K, D161L, D161M, D161Q, D161R,D161W, A162I, A162L, A162T, A162V, A162Y, Q163G, Q163L, Q163M, Q163S,P164A, P164C, P164D, P164M, P164N, P164R, P164V, P164W, F165D, F165E,F165G, F165H, F165I, F165K, F165L, F165M, F165R, F165S, F165T, F165V,F165Y, I166F, I166L, I166M, I166V, A167C, A167M, A167R, A167T, D168G,D168P, D168R, W169E, W169K, W169Q, M170F, M170H, M170L, M170T, M170V,M170Y, A171E, A171F, A171V, A171W, K172A, K172M, Q173N, Q175I, P176H,P176K, P176N, P176W, L177M, L177T, V178T, V178W, N179G, N179H, N179R,N179T, N179V, N179Y, H180E, H180G, H180R, H180V, H180W, D181A, D181H,D181I, D181L, D181P, D181R, D181W, S182A, S182G, S182K, S182L, S182P,and/or S182R, wherein the numbers in the substitution mutationdesignations refer to amino acid positions of SEQ ID NO:31. Theincreased substrate specificity for, and/or activity with respect to C₁₀substrates can be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₂ substrates (i.e., substrates, the carbonchains of which are 12 carbons in length), and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding to residues 10-25, 35-85,90-103, 110-143, 146-180 of SEQ ID NO:31. The increased substratespecificity for, and/or activity with respect to C12 substrates can bemeasured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent) is provided, which has an increasedsubstrate specificity for, and/or activity (e.g., catalytic rate) withrespect to C₁₂ substrates, and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated at one or moreamino acid positions corresponding one or more residues of SEQ ID NO:31selected from 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 35, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80,81, 82, 83, 84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 114,115, 116, 117, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 133, 134, 136, 137, 140, 141, 142, 145, 149, 152, 153, 155, 156,158, 159, 160, 161, 162, 163, 164, 166, 167, 168, 169, 170, 172, 173,174, 175, 176, 177, 179, 180, 181, and/or 182. The increased substratespecificity for, and/or activity with respect to C12 substrates can bemeasured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₂ substrates, and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated with oneor more substitutions selected from A1Q, A1S, A1V, D2E, D2K, D2P, D2W,T3R, T3W, L4A, L4Y, L5F, L5G, L5S, L5Y, I6T, I6V, L7A, L7C, L7M, L7N,L7S, L7T, L7V, L7Y, D9N, L11M, S12A, S12I, S12V, A13C, A13G, A13H, A13I,A13L, A13N, A13T, A13W, G14F, G14I, G14K, G14M, G14V, Y15A, Y15C, Y15D,Y15E, Y15G, Y15I, Y15L, Y15M, Y15N, Y15Q, Y15R, Y155, Y15V, R16D, R16E,R16G, R16H, R16I, R16L, R16N, R16P, R165, R16T, R16V, R16W, M17A, M17C,M17G, M17K, M17N, M17P, M17Q, M17R, M175, M17T, S18M, S18N, A19L, 520A,S20C, 520D, 520G, 520L, 520T, 520W, A21I, A21L, A21P, A21Y, A22F, A22L,A22M, A22N, A22R, A22Y, P24G, P24V, A25D, A25E, A25L, A25N, A25Q, A25R,A25V, L26D, L26E, L26F, L26G, L26H, L26I, L26K, L26N, L26R, L26S, L26W,L26Y, L27A, L27C, L27F, L27M, L27W, L27Y, N28R, N28W, D29P, K30P, W31E,W31N, T35L, T35Y, V37F, V37S, V37W, V38D, V38F, V38G, V38P, N39A, N39C,N39E, N39G, N39Q, N39W, A40D, A40L, A40M, A40P, A40V, A40Y, S41C, S41T,I42A, I42C, I42D, 142E, I42G, I42K, I42L, I42M, I42P, I42S, I42T, I42W,I42Y, S43A, S43D, S43E, S43F, S43G, S43H, S43L, S43M, S43N, S43R, S43T,S43V, G44C, G44E, G44H, G44K, G44L, G44N, G44Q, G44R, G44S, D45A, D45C,D45E, D45F, D45H, D45I, D45K, D45L, D45M, D45P, D45Q, D45S, D45T, D45V,D45W, T46A, T46C, T46D, T46G, T46K, T46N, T46R, T46S, S47P, S47Q, Q48E,Q48V, Q48W, Q48Y, Q49A, Q49C, Q49D, Q49E, Q49G, Q49H, Q49I, Q49K, Q49L,Q49M, Q49P, Q49R, Q49S, Q49V, Q49W, Q49Y, G50A, G50C, G50F, G50I, G50K,G50L, G50M, G50N, G50P, G50Q, G50R, G50S, G50T, G50Y, L51A, L51D, L51N,L51T, L51V, L51W, A52C, A52M, A52P, A52W, R53A, R53C, R53D, R53E, R53F,R53G, R53I, R53K, R53L, R53N, R535, R53T, R53V, R53W, R53Y, L54A, L54C,L54E, L54F, L54G, L54M, L54N, L545, L54W, L54Y, P55Y, L57A, L57C, L57F,L57K, L57P, L57Q, L57R, L57Y, L58A, L58D, L58E, L58G, L58H, L58N, L58R,L585, L58W, L58Y, Q60P, H61D, H61G, H61P, Q62P, Q62W, P63I, P63L, P63N,P63S, P63T, P63V, P63W, R64F, R64P, R64W, R64Y, W65A, W65E, W65G, W65K,W65M, W65N, W65V, V66M, V66S, L67A, L67T, V68A, V68L, V68M, V68S, V68T,E69A, E69C, E69D, E69G, E69H, E69K, E69L, E69M, E69N, E69P, E69V, E69Y,L70A, L70C, L70E, L70F, L70H, L70I, L70K, L70Q, L70S, L70T, L70V, G71A,G72A, N73G, N73H, N73L, N73R, N73S, N73T, D74E, D74G, L76I, L76M, L76W,R77C, R77D, R77E, R77G, R77K, R77L, R77Q, R77S, R77V, R77W, G78D, F79P,Q80G, Q80M, Q80S, Q80Y, P81A, P81E, P81K, P81L, P81M, P81W, P81Y, Q82F,Q82V, Q82W, Q82Y, Q83A, T84E, T84R, T84W, Q86A, Q86T, T87E, T87G, T87L,L88C, R89L, R89P, Q90N, Q90P, Q90W, I91G, I91M, I91S, I91V, I91Y, L92A,L92C, L92G, L92H, L92N, L92S, L92T, L92V, L92Y, Q93A, Q93G, Q93H, Q93I,Q93P, Q93Y, D94P, V95F, V95G, V95L, V95N, V95Q, V95T, V95W, K96A, K96L,K96P, K96Y, A97K, A97P, A98L, A98P, A98V, A98W, A98Y, N99C, N99D, N99G,N99L, N99M, N99P, N99Q, N99R, N99W, N99Y, A100D, A100E, A100G, A100H,A100I, A100K, A100L, A100Q, A100R, A100V, A100W, A100Y, E101G, E101L,E101M, E101P, E101S, E101T, E101V, P102E, P102F, P102H, P102L, P102Q,P102R, P102S, P102W, P102Y, L103E, L103K, L103N, L103Q, L103R, L104C,L104P, L104S, L104W, M105C, M105E, M105G, M105V, Q106A, Q106C, Q106G,Q106K, Q106R, Q106S, Q106T, 1107C, 1107E, 1107K, I107L, 1107M, 1107S,1107V, R108F, R108W, L109M, AMC, A111Q, A111W, N112A, N112G, N112W,Y113A, Y113D, Y113G, Y113I, G114K, G114L, G114M, G114Y, R115A, R115C,R115E, R115G, R115N, R115S, R115Y, R116H, R116W, Y117C, Y117H, Y117I,Y117L, Y117M, Y117N, Y117S, Y117T, Y117V, E119C, E119F, E119K, E119M,E119R, E119W, E119Y, A120D, A120G, A120I, A120T, A120W, S122F, S122I,S122L, S122M, S122V, S122W, S122Y, A123C, A123F, A123H, A123L, A123R,A123T, A123V, A123W, A123Y, I124G, I124H, I124K, I124L, I124R, I124S,I124Y, Y125F, Y125R, P126C, P126F, P126H, P126Y, K127I, K127P, L128A,L128S, L128T, A129H, A129I, A129K, A129N, A129W, A129Y, K130P, E131A,E131C, E131F, E131G, E131K, E131L, E131N, E131V, E131W, D133K, V134D,V134E, V134K, V134N, V134Q, V134R, V134W, V134Y, L136A, L136D, L136E,L136F, L136G, L136H, L136K, L136N, L136P, L136Q, L136R, L136S, L136T,L137E, L137G, L137H, L137P, L137Q, L137S, L137Y, F140M, M141A, M141C,M141L, M141P, E142C, Y145E, Q149L, Q152A, Q152D, Q152E, Q152H, Q152K,Q152R, Q152Y, D153K, G155F, G155W, G155Y, I156C, I156F, I156M, I156V,P158A, P158G, N159G, N159Q, N159T, N159V, R160A, R160D, R160E, R160G,R160H, R160N, R160Q, R160S, R160W, D161I, D161K, D161L, D161M, D161N,D161Q, D161W, A162G, Q163A, Q163C, Q163G, Q163L, Q163M, Q163S, Q163T,P164C, P164M, I166L, I166V, A167C, A167E, A167F, A167G, A167K, A167L,A167N, A167Q, A167R, A167T, A167V, A167Y, D168G, D168H, D168L, D168R,D168V, D168W, W169A, W169D, W169E, W169G, W169K, W169Q, W1695, M170F,M170G, M170N, M170Q, M1705, M170V, M170W, K172M, K172P, Q173N, L174A,L174F, L174G, L174T, L174W, Q175I, P176H, P176K, P176L, P176N, P176W,L177D, L177G, N179H, N179R, N179Y, H180A, H180G, D181H, D181I, D181L,D181R, D181W, S182K, S182L, S182P, and/or S182R, wherein the numbers inthe substitution mutation designations refer to amino acid positions ofSEQ ID NO:31. The increased substrate specificity for, and/or activitywith respect to C₁₂ substrates can be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₄ substrates (i.e., substrates, the carbonchains of which are 14 carbons in length), and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding to residues 5-20, 35-58,65-80, 83-90, 110-130, 140-145, 155-160, 165-180 of SEQ ID NO:31. Theincreased substrate specificity for, and/or activity with respect to C14substrates can be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₄ substrates, and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding one or more residues of SEQID NO:31 selected from 1, 4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 20,21, 22, 23, 25, 26, 28, 29, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 66, 68, 69, 70, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 91, 92, 93,95, 96, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 131, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 147, 148,151, 152, 153, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 173, 174, 175, 176, 178, 179, 180, 181, and/or182. The increased substrate specificity for, and/or activity withrespect to C14 substrates can be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has anincreased substrate specificity for, and/or activity (e.g., catalyticrate) with respect to C₁₄ substrates, and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated with oneor more substitutions selected from A1S, L4S, L4Y, L5H, L5Y, L7C, L7M,L7N, L7S, L7T, L7Y, G8S, D9N, D9T, L11C, L11I, L11M, L11Q, Lily, S12I,S12L, S12M, S12T, S12V, A13H, A13I, A13L, A13T, A13V, G14F, G14I, G14R,G14T, G14V, Y15A, Y15C, Y15D, Y15E, Y15G, Y15I, Y15L, Y15M, Y15N, Y15Q,Y15R, Y15S, Y15V, R16G, R16N, R16P, R16W, M17C, M17D, M17G, M17K, M17N,M17P, M17R, M17S, M17T, S20A, S20D, S20G, S20L, S20T, S20W, A21G, A22L,A22N, A22Y, W23Y, A25E, A25N, A25Q, A25V, L26C, L26F, L26H, L26Q, L26V,L26Y, N28K, N28P, D29V, S33F, S36H, V37H, V37Q, V38F, N39F, N39M, N39Q,N39V, N39W, N39Y, A40G, A40P, A40T, A40V, S41P, S41T, I42A, I42D, 142E,I42G, I42L, I42M, I42P, I42S, I42T, 142W, I42Y, S43A, S43D, S43E, S43F,S43G, S43H, S43L, S43M, S43N, S43T, S43V, S43W, G44A, G44C, G44E, G44F,G44H, G44K, G44L, G44M, G44N, G44Q, G44R, G44S, G44W, G44Y, D45A, D45C,D45E, D45F, D45G, D45H, D45M, D45P, D45Q, D45S, D45T, D45V, D45W, T46A,T46C, T46D, T46G, T46K, T46N, T46S, T46W, S47E, S47P, S47Q, S47W, S47Y,Q48C, Q48F, Q48I, Q48M, Q48V, Q48W, Q48Y, Q49A, Q49C, Q49D, Q49E, Q49G,Q49H, Q49I, Q49K, Q49L, Q49M, Q49P, Q49R, Q49S, Q49V, Q49W, Q49Y, G50A,G50C, G50E, G50F, G50I, G50K, G50L, G50M, G50N, G50P, G50Q, G50R, G50S,G50T, G50W, G50Y, L51A, L51C, L51D, L51S, L51V, A52H, A52I, A52L, A52M,A52P, A52R, A52V, A52W, A52Y, R53A, R53C, R53D, R53E, R53F, R53G, R53I,R53K, R53L, R53N, R53S, R53T, R53V, R53W, R53Y, L54W, L54Y, A56R, A56W,A56Y, L57F, L58F, L58I, L58Y, V66I, V68L, E69A, E69C, E69D, E69F, E69G,E69H, E69K, E69L, E69M, E69N, E69Q, E69S, E69V, E69Y, L70A, L70C, L70E,L70F, L70H, L70Q, L70S, L70T, L70V, L70W, G72A, G72C, G72P, G72S, N73A,N73C, N73G, N73H, N73I, N73L, N73P, N73R, N73S, N73T, N73V, N73W, D74E,D74G, G75A, G75C, G75D, G75E, G75F, G75I, G75K, G75L, G75M, G75N, G75P,G75T, G75W, G75Y, L76A, L76C, L76D, L76E, L76F, L76G, L76I, L76K, L76M,L76N, L76P, L76Q, L76R, L76T, L76V, L76W, R77A, R77C, R77D, R77E, R77F,R77G, R77H, R77K, R77L, R77N, R77Q, R77S, R77V, R77W, G78P, F79M, F79P,F79V, Q80A, Q80G, Q80L, Q80M, Q80S, Q80W, Q80Y, P81A, P81E, P81K, P81L,P81M, P81W, P81Y, Q82F, Q82I, Q82N, Q82P, Q82V, Q82W, Q82Y, Q83A, T84S,E85D, Q86A, Q86T, Q86V, Q86W, T87A, T87C, T87E, T87F, T87G, T87H, T87L,T87M, T87S, T87V, T87W, R89H, R89T, R89V, R89W, I91L, I91V, I91Y, L92V,Q93A, Q93G, Q93H, Q93I, Q93P, Q93Y, V95L, V95M, V95T, V95W, K96A, K96L,K96P, K96Y, A97W, A98K, A98L, A98W, N99G, N99L, N99P, N99Q, N99R, N99Y,A100G, A100H, A100I, A100K, A100L, A100M, A100R, A100T, A100V, A100Y,E101G, E101L, E101M, E101S, E101T, E101V, P102S, M105A, M105C, M105E,M105G, M105I, M105L, M105V, Q106A, Q106C, Q106D, Q106G, Q106H, Q106K,Q106L, Q106M, Q106R, Q106S, Q106T, Q106V, Q106W, Q106Y, 1107C, 1107E,1107G, I107L, 1107M, I107Q, 1107V, R108A, R108C, R108D, R108F, R108I,R108L, R108S, R108V, R108W, R108Y, L109C, L109M, L109Q, L109T, L109V,L109Y, P110A, P110E, P110H, P110N, P110R, P110V, A111C, A111L, A111Q,A111R, A111V, A111W, N112A, N112F, N112G, N112I, N112L, N112P, N112V,N112W, N112Y, Y113A, Y113D, Y113G, Y113I, Y113M, Y113W, G114F, G114K,G114L, G114M, G114W, G114Y, R115A, R115C, R115E, R115G, R115I, R115N,R115P, R115Q, R115S, R115V, R115W, R115Y, R116C, R116H, R116T, R116V,R116W, Y117C, Y117H, Y117I, Y117L, Y117M, Y117N, Y117S, Y117W, N118A,N118C, N118E, N118G, N118H, N118I, N118L, N118M, N118P, N118Q, N118T,N118V, N118W, E119C, E119D, E119F, E119K, E119M, E119P, E119R, E119T,E119W, E119Y, A120D, A120G, A120I, A120L, A120T, A120W, F121A, F121C,F121D, F121E, F121K, F121L, F121M, F121P, F121Q, F121R, F121S, F121V,F121Y, S122A, S122C, S122D, S122E, S122F, S122G, S122I, S122L, S122M,S122P, S122V, S122W, S122Y, A123C, A123E, A123F, A123H, A123L, A123T,A123V, A123W, A123Y, I124A, I124C, I124G, I124L, I124Y, Y125C, Y125F,Y125G, Y125I, Y125L, Y125P, Y125Q, Y125R, Y125S, Y125T, Y125V, P126C,P126H, P126Y, E131I, E131L, D133K, D133Y, V134S, L136C, L136M, L136Q,L136S, L137P, P138E, P138R, P138T, F139M, F140M, M141A, M141C, M141L,M141P, E142A, E142C, E142L, E142M, E142N, E142P, E142Q, E142S, E142Y,E143I, E143P, K147R, P148W, M151I, M151Q, M151V, Q152A, Q152K, Q152S,D153I, D153K, D153M, D153W, G155F, G155H, G155W, G155Y, I156C, I156F,I156M, I156Q, I156R, I156S, I156V, P158A, P158G, P158S, N159G, N159T,R160A, R160G, R160H, R160N, R160W, D161G, D161I, D161K, D161L, D161M,D161N, D161Q, D161R, D161S, D161V, D161W, A162G, Q163G, Q163L, Q163M,Q163S, P164A, P164C, P164K, P164L, P164M, P164N, P164R, P164T, P164W,F165G, F165H, F165S, F165W, F165Y, I166L, I166V, A167T, D168A, D168G,D168H, D168P, D168R, D168T, W169A, W169E, W169K, W169M, W169Q, W169R,W169S, W169T, W169V, M170A, M170F, M170V, A171I, Q173N, Q173W, Q173Y,L174Q, L174W, Q175I, Q175Y, P176H, P176K, P176L, P176R, P176W, P176Y,V178A, V178T, V178W, N179H, N179R, N179T, N179V, N179Y, H180G, H180R,H180S, H180W, D181A, D181H, D181I, D181L, D181Q, D181R, D181S, D181W,S182A, S182E, S182G, S182I, S182K, S182L, S182P, S182Q, S182R, and/orS182T, wherein the numbers in the substitution mutation designationsrefer to amino acid positions of SEQ ID NO:31. The increased substratespecificity for, and/or activity with respect to C14 substrates can bemeasured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has apreference for ester substrates (e.g., acyl-PNP) over thioestersubstrates (e.g., acyl-CoA), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated at one or moreamino acid positions corresponding to residues selected from 95, 96, 97,98, 99, 100, 101, 102, 104, 105, 106, 107, 108, 109, and/or 110 of SEQID NO:31. The preference for ester substrates over thioester substratescan be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has apreference for ester substrates (e.g., acyl-PNP) over thioestersubstrates (e.g., acyl-CoA), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated with one or moresubstitutions selected from V95L, V95M, V95T, K96A, K96L, K96W, K96Y,A97F, A97K, A97S, A97T, A97W, A98E, A98F, A98K, A98L, A98Q, A98W, N99Y,A100K, A100V, E101L, P102S, L104C, M105F, Q106A, Q106C, Q106T, Q106Y,1107A, 1107C, 1107G, I107L, I107M, I107Q, 1107V, R108A, R108C, R108D,R108F, R1081, R108L, R108S, R108V, R108W, R108Y, L109M, L109V, P110A,P110F, P110H, P110N, P110V, and/or P110W, wherein the numbers in thesubstitution mutation designations refer to amino acid positions of SEQID NO:31. The preference for ester substrates over thioester substratescan be measured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has apreference for thioester substrates (e.g., acyl-CoA) over estersubstrates (e.g., acyl-PNP), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated at one or moreamino acid positions corresponding to residues selected from 95, 96, 97,101, 102, 103, 104, 105, 107, 109, and/or 110 of SEQ ID NO:31. Thepreference for thioester substrates over ester substrates can bemeasured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which has apreference for thioester substrates (e.g., acyl-CoA) over estersubstrates (e.g., acyl-PNP), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated with one or moresubstitutions selected from V95E, V95I, V95W, V95Y, K96P, A97E, A97M,E101P, P102D, P102K, P102Y, L103E, L103K, L103N, L104A, L104D, L104E,L104N, L104Q, L104W, L104Y, M105W, 1107E, 1107K, 1107P, L109A, L109C,L109D, L109E, L109G, L109K, L109N, L109P, L109Q, L109S, L109T, L109Y,and/or P110R, wherein the numbers in the substitution mutationdesignations refer to amino acid positions of SEQ ID NO:31. Thepreference for thioester substrates over ester substrates can bemeasured in vitro and/or in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofproducing an increased proportional or percentage yield of fatty estersover other non-fatty ester products (e.g., free fatty acids and/or fattyacid derivatives other than fatty esters), and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding to residues of SEQ ID NO:31selected from 1-14, 22-29, 33-58, 65-100, 103-109, 114-117, 119-121,127-136, 139-144, 150-151, 155-170, and/or 173-174. The increasedproportional or percentage yield of fatty esters over other products(e.g., fatty acid derivatives other than fatty esters) can be observedor determined in vitro and/or in vivo. Preferably, the increasedproportional or percentage yield of fatty esters over other products isdetermined in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofproducing an increased proportional or percentage yield of fatty estersover other products (e.g., free fatty acids and/or fatty acidderivatives other than fatty esters), and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated at oneor more amino acid positions corresponding to residues of SEQ ID NO:31selected from 1, 2, 4, 5, 6, 7, 8, 12, 13, 14, 22, 23, 24, 25, 26, 28,29, 33, 34, 35, 36, 37, 38, 39, 40, 41, 44, 45, 46, 47, 49, 50, 53, 58,65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79, 81, 84, 86, 87, 88,89, 90, 91, 92, 93, 95, 96, 99, 100, 103, 104, 105, 106, 107, 108, 109,114, 115, 117, 119, 120, 121, 127, 128, 129, 131, 132, 134, 135, 136,139, 141, 142, 143, 144, 150, 151, 155, 156, 158, 159, 160, 161, 162,163, 164, 165, 166, 169, 170, 173, and/or 174. The increasedproportional or percentage yield of fatty esters over other products(e.g., fatty acid derivatives other than fatty esters) can be observedor determined in vitro and/or in vivo. Preferably, the increasedproportional or percentage yield of fatty esters over other products(e.g., fatty acid derivatives other than fatty esters) is determined invivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofproducing an increased proportional or percentage yield of fatty estersover other products (e.g., free fatty acids and/or fatty acidderivatives other than fatty esters), and which is a variant of aprecursor thioesterase that comprises an analogous sequence to SEQ IDNO:31 in FIG. 58, wherein the precursor thioesterase is mutated with oneor more substitutions selected from A1R, D2H, D2R, L4G, L4M, L5Q, I6A,I6L, L7E, G8A, S12N, A13I, A13L, A13S, A13T, A13W, A13Y, G14K, G14R,G14S, G14T, A22D, A22E, A22H, A22Y, W23Y, P24C, P24G, P24T, A25P, L26C,L26D, L26E, L26G, L26N, N28A, N28M, D29V, S33G, S33M, K34A, K34H, K34M,T35G, T35M, S36A, V37A, V37G, V37H, V37S, V38D, V38G, V38P, N39E, N39Q,N39R, A40M, A40P, S41T, G44F, G44Y, D45P, D45Q, T46W, S47F, Q49I, G50A,G50K, G50M, G50S, R53S, L58D, L58M, L58R, W65L, L67G, V68G, V68M, V68N,E69P, E69Q, L70A, L70E, L70H, G71C, G72A, N73C, N73G, N73L, N73R, N73T,N73V, D74C, D74S, D74W, G75A, G75K, G75L, G75M, L76A, L76F, L76G, L76I,L76M, L76N, L76T, L76W, R77G, F79A, F79M, F79P, P81E, P81W, T84F, T84H,T84Y, Q86P, Q86W, T87M, T87S, T87W, L88C, L88F, L88G, L88H, L88Y, R89G,Q90P, Q90W, I91M, I91S, L92C, L92G, Q93F, Q93P, V95A, V95D, V95E, V95L,V95M, K96P, N99L, N99M, N99S, A100D, A100K, A100L, A100M, A100V, A100Y,L103A, L104A, L104C, L104P, L104Q, L104W, M105A, Q106A, Q106C, Q106T,Q106W, 1107C, 1107M, R108E, L109F, L109M, G114F, R115W, Y117P, E119D,E119P, A120P, F121A, F121C, F121W, K127P, L128F, A129L, A129Y, E131A,F132P, V134P, P135A, L136A, F139M, M141A, M141P, E142A, E143P, V144A,W150D, W150E, M151S, G155V, I156K, I156M, P158A, P158G, P158Q, P158S,N159E, N159I, R160H, R1601, R160K, D161G, A162T, A162Y, Q163A, Q163C,Q163E, Q163G, Q163I, Q163M, Q163S, Q163T, Q163V, P164C, F165D, F165S,I166A, I166L, W169M, M170E, M170G, M170N, M170S, Q173P, and/or L174A,wherein the numbers in the substitution mutation designations refer toamino acid positions of SEQ ID NO:31. The increased proportional orpercentage yield of fatty esters over other products (e.g., fatty acidderivatives other than fatty esters) can be observed or determined invitro and/or in vivo. Preferably, the increased proportional orpercentage yield of fatty esters over other products (e.g., fatty acidderivatives other than fatty esters) is determined in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofproducing a decreased proportional or percentage yield of fatty estersover other products (e.g., free fatty acids and/or fatty acidderivatives other than fatty esters) when fatty ester production isundesirable, and which is a variant of a precursor thioesterase thatcomprises an analogous sequence to SEQ ID NO:31 in FIG. 58, wherein theprecursor thioesterase is mutated at one or more amino acid positionscorresponding to residues of SEQ ID NO:31 selected from 3, 5, 15-18,27-42, 46, 57-68, 77-78, 95-106, 121-123, 152-154, 167, and/or 175-182.The decreased proportional or percentage yield of fatty esters overother products (e.g., fatty acid derivatives other than fatty esters)can be observed or determined in vitro and/or in vivo. Preferably, thedecreased proportional or percentage yield of fatty esters over otherproducts (e.g., fatty acid derivatives other than fatty esters) isdetermined in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofproducing a decreased proportional or percentage yield of fatty estersover other products (e.g., free fatty acids and/or fatty acidderivatives other than fatty esters) when fatty ester production isundesirable, and which is a variant of a precursor thioesterase thatcomprises an analogous sequence to SEQ ID NO:31 in FIG. 58, wherein theprecursor thioesterase is mutated at one or more amino acid positionscorresponding to residues of SEQ ID NO:31 selected from 3, 5, 15, 16,18, 27, 28, 33, 34, 35, 36, 37, 38, 40, 42, 46, 57, 59, 60, 62, 65, 68,77, 78, 95, 96, 97, 98, 99, 100, 102, 103, 105, 106, 121, 123, 152, 153,154, 167, 175, 176, 178, 179, 180, 181, and/or 182. The decreasedproportional or percentage yield of fatty esters over other products(e.g., fatty acid derivatives other than fatty esters) can be observedor determined in vitro and/or in vivo. Preferably, the decreasedproportional or percentage yield of fatty esters over other products(e.g., fatty acid derivatives other than fatty esters) is determined invivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofproducing a decreased proportional or percentage yield of fatty estersover other products (e.g., free fatty acids and/or fatty acidderivatives other than fatty esters) when production of fatty esters isundesirable, and which is a variant of a precursor thioesterase thatcomprises an analogous sequence to SEQ ID NO:31 in FIG. 58, wherein theprecursor thioesterase is mutated with one or more substitutionsselected from T3E, T3G, T3K, T3L, L5C, L5G, Y15A, Y15L, Y15Q, Y15R,Y15V, R16D, R16E, R16G, R16I, R16V, S18E, L27V, N28G, N28I, S33I, S33R,K34R, T35F, T35K, T35L, T35Q, T35V, S36F, S36I, S36L, S36W, V37L, V38E,V38F, V38K, V38L, A40D, A40G, I42T, T46L, L57A, L57F, L57G, L57H, L57K,L57N, L57P, L57R, L57S, L57T, L57V, L57W, L57Y, K59V, Q60E, Q60P, Q62G,W65V, V68L, R77L, G78M, V95F, V95N, K96C, K96L, K96N, K96Q, K96R, K96Y,A97E, A97F, A97R, A97W, A98E, N99A, N99D, A100S, P102I, L103Q, L103W,M105L, Q106G, Q106H, Q106K, Q106S, Q106V, F121P, A123E, Q152D, Q152E,Q152F, Q152H, Q152I, Q152K, Q152L, Q152S, Q152T, Q152Y, D153P, D153V,D154E, A167V, Q175L, P176D, V178K, N179H, N179W, H180E, H180L, H180P,H180R, D181C, D181E, S182K, S182L, S182N, S182R, S182T, and/or S182V,wherein the numbers in the substitution mutation designations refer toamino acid positions of SEQ ID NO:31. The decreased proportional orpercentage yield of fatty esters over other products (e.g., fatty acidderivatives other than fatty esters) can be observed or determined invitro and/or in vivo. Preferably, the decreased proportional orpercentage yield of fatty esters over other products (e.g., fatty acidderivatives other than fatty esters) is determined in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofincreased and/or improved production of one or more fatty acidderivatives, and which is a variant of a precursor thioesterase thatcomprises an analogous sequence to SEQ ID NO:31 in FIG. 58, wherein theprecursor thioesterase is mutated at one or more amino acid positionscorresponding to residues of SEQ ID NO:31 selected from 2, 4, 11-22,25-31, 37-45, 49-58, 63-80, 84-130, 136-146, and/or 150-174. Anexemplary fatty acid derivative that is produced accordingly is a freefatty acid. The increased and/or improved production of fatty acidderivatives can be measured in vitro and/or in vivo. Preferably, theincreased and/or improved production of fatty acid derivatives ismeasured in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofincreased and/or improved production of one or more fatty acidderivatives, and which is a variant of a precursor thioesterase thatcomprises an analogous sequence of SEQ ID NO:31 in FIG. 58, wherein theprecursor thioesterase is mutated at one or more amino acid positionscorresponding to residues of SEQ ID NO:31 selected from 2, 4, 11, 12,13, 14, 15, 16, 17, 19, 21, 22, 25, 26, 27, 28, 29, 30, 31, 37, 39, 41,42, 43, 44, 45, 49, 50, 51, 53, 54, 58, 63, 65, 66, 67, 68, 69, 70, 71,73, 74, 75, 76, 77, 78, 79, 80, 84, 87, 88, 90, 91, 92, 93, 94, 95, 96,97, 98, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,115, 117, 118, 119, 120, 121, 122, 124, 127, 128, 129, 130, 136, 137,138, 139, 140, 141, 143, 144, 145, 146, 150, 151, 152, 154, 155, 156,158, 162, 163, 166, 167, 169, 170, 173, and/or 174. An exemplary fattyacid derivative that is produced accordingly is a free fatty acid. Theincreased and/or improved production of a fatty acid derivative can bemeasured in vitro and/or in vivo. Preferably, the increased and/orimproved production of a fatty acid derivative is measured in vivo.

In one embodiment of the invention, a mutant thioesterase (or anaturally-occurring equivalent thereof) is provided, which is capable ofincreased and/or improved production of one or more fatty acidderivatives, and which is a variant of a precursor thioesterase thatcomprises an analogous sequence to SEQ ID NO:31 in FIG. 58, wherein theprecursor thioesterase is mutated with one or more substitutionsselected from: D2L, D2P, D2R, L5G, L11I, S12N, S12T, A13N, G14C, G14P,G14S, G14T, G14V, Y15C, Y15I, Y15V, R16T, M17D, M17E, M17N, M17R, M17S,M17V, A19C, A21G, A22L, A22R, A22T, A25P, L26D, L26G, L26W, L27C, L27F,L27W, L27Y, N28I, N28P, D29P, K30P, W31D, W31G, W31N, W31P, W31R, W31S,W31T, V37Y, N39P, S41C, I42D, I42G, S43E, G44K, G44R, G44W, D45G, Q49E,G50A, G50K, G50M, G50Q, L51D, L51T, R53A, R53G, R53L, R53N, R535, R53V,L54E, L54F, L54G, L54N, L545, L54W, L58R, P63G, P63M, P63N, P63T, P63W,W65E, W65G, V66G, V66S, L67T, V68S, E69F, E69V, L70C, L70F, L70Q, L705,L70T, L70V, G71A, N73G, N73L, D74A, D74C, G75A, G75C, G75F, G75R, G75W,L76I, R77A, R77C, R77D, R77F, R77G, R77H, R77K, R77L, R77N, R77Q, R77S,R77W, G78D, G78E, F79K, Q80G, T84H, T84N, T84Q, T87A, T87F, T87H, T87W,L88A, L88C, L88H, Q90N, Q90W, I91G, I91L, I91M, I91S, L92G, L92N, L92Q,L92S, L92T, L92Y, Q93P, D94P, V95F, V95N, V95Q, K96P, A97C, A97P, A98P,A98V, A100D, A100E, A100Q, A100Y, P102L, P102Q, P102R, L103E, L103K,L104A, L104Q, L104W, L104Y, M105C, M105E, M105F, M105L, Q106D, Q106G,Q106L, Q106V, Q106W, Q106Y, 1107A, 1107C, 1107E, 1107G, I107K, I107L,I107Q, I107S, I107T, R108G, L109F, L109V, L109Y, P110A, P110E, P110F,P110G, P110H, P110N, P110S, P110V, A111Y, N112F, N112P, Y113D, Y113E,Y113P, R115W, Y117A, Y117D, Y117E, Y117G, Y117P, Y117Q, N118F, E119P,A120P, F121C, F121L, F121M, F121N, F121Q, F121R, F121V, F121W, F121Y,S122D, S122F, S122L, S122P, S122W, S122Y, I124A, I124G, I124H, I124K,I124R, K127P, L1285, A129I, A129W, A129Y, K130P, L136A, L136D, L136E,L136G, L136K, L136N, L136P, L136Q, L1365, L136T, L137A, L137C, L137H,L137K, L137Q, L1375, L137Y, P138F, F139L, F139M, F140C, F140I, F140L,F140M, F140V, M141T, E143P, V144H, Y145I, L146G, L146P, W150G, W150I,W150V, M151F, M151L, M151R, M1515, M151T, M151W, Q152N, Q152V, Q152Y,D154C, D154E, G155I, I156C, I156K, I156T, I156V, P158G, P158T, A162T,Q163A, Q163C, Q163E, Q163I, Q1635, Q163T, Q163V, I166C, A167E, A167F,A167L, A167N, A167R, A167V, A167Y, W169K, M170N, M1705, Q173D, L174A,L174T, and/or L174W, wherein the numbers in the substitution mutationdesignations refer to amino acid positions of SEQ ID NO:31. An exemplaryfatty acid derivative produced accordingly is a free fatty acid. Theincreased and/or improved production of a fatty acid derivative can bemeasured in vitro and/or in vivo. Preferably, the increased and/orimproved production of a fatty acid derivative is measured in vivo.

In one embodiment, a mutant thioesterase (or a naturally-occurringequivalent thereof) is provided, which is capable of producing anincreased proportional or percentage yield of short-chain (e.g., C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄) fatty acid derivatives (e.g., short-chain fattyacids, short-chain fatty esters, short-chain fatty alcohols, etc.) vs.other products (e.g., non-short-chain fatty acid derivatives, including,for example, long-chain (e.g., C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀) fattyacids, long-chain fatty esters, long-chain fatty alcohols, etc.), andwhich is a variant of a precursor thioesterase that comprises ananalogous sequence to SEQ ID NO:31 in FIG. 58, wherein the precursorthioesterase is mutated at one or more amino acid positionscorresponding to one or more residues of SEQ ID NO:31 selected from 13,16-17, 25-38, 55-67, 78-98, 105-119, 122, 126, 132-145, 153, and/or161-182. An exemplary short-chain fatty acid derivative is a C₁₂ fattyacid derivative. An alternative short-chain fatty acid derivative is aC₁₄ fatty acid derivative. In certain circumstances, the increasedproportional or percentage yield of short-chain fatty acid derivativecan be correlated to a decreased proportional yield of long-chain fattyacid derivatives. The increased proportional or percentage yield ofshort-chain fatty acid derivatives and/or the corresponding decreasedproportional or percentage yield of long-chain fatty acid derivativescan be measured in vitro or in vivo. Preferably, the increasedproportional yield of short-chain fatty acid derivatives or thecorresponding decreased proportional or percentage yield of long-chainfatty acid derivatives is measured in vivo.

In one embodiment, a mutant thioesterase (or a naturally-occurringequivalent thereof) is provided, which is capable of producing anincreased proportional or percentage yield of short-chain (e.g., C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄) fatty acid derivatives (e.g., short-chain fattyacids, short-chain fatty esters, short-chain fatty alcohols, etc.) vs.other products (e.g., non-short-chain fatty acid derivatives, including,for example, long-chain fatty acids, long-chain fatty esters, long-chainfatty alcohols, etc.), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated at one or moreamino acid positions corresponding to one or more residues of SEQ IDNO:31 selected from 13, 16, 17, 25, 29, 31, 35, 36, 38, 55, 57, 58, 59,61, 62, 63, 64, 65, 66, 67, 78, 79, 82, 83, 84, 85, 86, 87, 89, 90, 93,94, 95, 96, 97, 98, 105, 106, 108, 111, 113, 114, 117, 119, 122, 126,132, 135, 136, 139, 142, 144, 145, 153, 161, 162, 165, 168, 173, 175,176, 178, 179, 180, 181, and/or 182. An exemplary short-chain fatty acidderivative is a C₁₂ fatty acid derivative. An alternative short-chainfatty acid derivative is a C₁₄ fatty acid derivative. In certaincircumstances, the increased proportional or percentage yield ofshort-chain fatty acid derivatives can be correlated to a decreasedproportional yield of long-chain fatty acid derivatives. The increasedproportional or percentage yield of short-chain fatty acid derivativesand/or the corresponding decreased proportional or percentage yield oflong-chain fatty acid derivatives can be measured in vitro or in vivo.Preferably, the increased proportional yield of short-chain fatty acidderivatives or the corresponding decreased proportional yield oflong-chain fatty acid derivatives is measured in vivo.

In one embodiment, a mutant thioesterase (or a naturally-occurringequivalent thereof) is provided, which is capable of producing anincreased proportional or percentage yield of short-chain (e.g., C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄) fatty acid derivatives (e.g., short-chain fattyacids, short-chain fatty esters, short-chain fatty alcohols, etc.) vs.other products (e.g., non-short-chain fatty acid derivatives including,for example, long-chain fatty acids, long-chain fatty esters, long-chainfatty alcohols, etc.), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated with one or moresubstitution selected from: A13V, R16A, M17T, A25S, D29M, W31L, T35Y,S36W, V38S, P55A, P55G, L57I, L58M, L58V, K59E, H61W, Q62M, P63V, R64M,W65L, V66C, L67C, L67M, G78F, G78M, G78R, G78T, G78V, F79K, F79Y, Q82A,Q82M, Q82R, Q83G, Q83K, T84M, T84V, E85A, E85C, E85G, E85Q, E85S, E85T,E85V, E85W, E85Y, Q86H, Q86Y, T87R, R89V, Q90L, Q93M, Q93N, Q93V, D94C,D94L, V95G, K96C, A97N, A97V, A98G, A98Y, M105I, Q106K, Q106R, R108W,A111E, A111N, A111S, A111W, A111Y, Y113A, Y113S, Y113V, G114K, G114Y,Y117R, E119M, E119Q, E119R, S122F, S122I, S122M, S122R, P126K, F132C,F132D, F132K, F132L, F132N, F132V, P135A, P135E, P135K, P135Q, L136H,F139L, E142W, V144Y, Y145A, Y145C, Y145D, Y145E, Y145G, Y1451, Y145L,Y145M, Y145N, Y145R, Y145S, Y145T, D153K, D153Q, D161K, A162I, F165K,D168W, Q1731, Q175M, P176Q, P176R, P176V, V178F, V178G, V178L, V178R,V178S, V178T, N179H, H180E, H180P, H180R, H180S, H180V, H180W, D181R,D181T, S182C, S182D, S182G, and/or S182R, wherein the numbers in thesubstitution mutation designations refer to amino acid positions of SEQID NO:31. An exemplary short-chain fatty acid derivative is a C₁₂ fattyacid derivative. An alternative short-chain fatty acid derivative is aC₁₄ fatty acid derivative. In certain circumstances, the increasedproportional or percentage yield of short-chain fatty acid derivativescan be correlated to a decreased proportional yield of long-chain fattyacid derivatives. The increased proportional or percentage yield ofshort-chain fatty acid derivatives and/or the corresponding decreasedproportional yield of long-chain fatty acid derivatives can be measuredin vitro or in vivo. Preferably, the increased proportional yield ofshort-chain fatty acid derivatives or the corresponding decreasedproportional yield of long-chain fatty acid derivatives is measured invivo.

In one embodiment, a mutant thioesterase (or a naturally-occurringequivalent thereof) is provided, which is capable of producing adecreased proportional or percentage yield of short-chain (e.g., C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄) fatty acid derivatives (e.g., short-chain fattyacids, short-chain fatty esters, short-chain fatty alcohols, etc.) vs.other products (e.g., non-short-chain fatty acid derivatives including,for example, long-chain (e.g., C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀) fattyacids, long-chain fatty esters, long-chain fatty alcohols, etc.), andwhich is a variant of a precursor thioesterase that comprises ananalogous sequence to SEQ ID NO:31 in FIG. 58, wherein the precursorthioesterase is mutated at one or more amino acid positionscorresponding to one or more residues of SEQ ID NO:31 selected from1-31, 36-81, 84-159, 162-177, and/or 181. An exemplary short-chain fattyacid derivative is a C₁₂ fatty acid derivative. An alternativeshort-chain fatty acid derivative is a C₁₄ fatty acid derivative. Incertain circumstances, the decreased proportional or percentage yield ofshort-chain fatty acid derivatives can be correlated to an increasedproportional yield of long-chain fatty acid derivatives. The decreasedproportional or percentage yield of short-chain fatty acid derivativesand/or the corresponding increased proportional yield of long-chainfatty acid derivatives can be measured in vitro or in vivo. Preferably,the decreased proportional yield of short-chain fatty acid derivativesor the corresponding increased proportional yield of short-chain fattyacid derivatives is measured in vivo.

In one embodiment, a mutant thioesterase (or a naturally-occurringequivalent thereof) is provided, which is capable of producing adecreased proportional or percentage yield of short-chain (e.g., C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄) fatty acid derivatives (e.g., short-chain fattyacids, short-chain fatty esters, short-chain fatty alcohols, etc.) vs.other products (e.g., non-short-chain fatty acid derivatives including,for example, long-chain fatty acids, long-chain fatty esters, long-chainfatty alcohols, etc.), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated at one or moreamino acid positions corresponding to one or more residues of SEQ IDNO:31 selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16,17, 18, 19, 21, 22, 23, 24, 26, 27, 30, 31, 36, 37, 38, 42, 44, 45, 46,47, 48, 50, 51, 52, 53, 54, 55, 57, 61, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 117, 118, 119, 120, 121, 122, 124, 125,127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 162, 163, 165, 166, 167, 168, 170, 171, 173, 174, 175, 176, 177,and/or 181. An exemplary short-chain fatty acid derivative is a C₁₂fatty acid derivative. An alternative short-chain fatty acid derivativeis a C₁₄ fatty acid derivative. In certain circumstances, the decreasedproportional or percentage yield of short-chain fatty acid derivativescan be correlated to an increased proportional yield of long-chain fattyacid derivatives. The decreased proportional or percentage yield ofshort-chain fatty acid derivatives and/or the corresponding increasedproportional yield of long-chain fatty acid derivatives can be measuredin vitro or in vivo. Preferably, the decreased proportional yield ofshort-chain fatty acid derivatives or the corresponding increasedproportional yield of short-chain fatty acid derivatives is measured invivo.

In one embodiment, a mutant thioesterase (or a naturally-occurringequivalent thereof) is provided, which is capable of producing adecreased proportional or percentage yield of short-chain (e.g., C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄) fatty acid derivatives (e.g., short-chain fattyacids, short-chain fatty esters, short-chain fatty alcohols, etc.) vs.other products (e.g., non-short-chain fatty acid derivatives including,for example, long-chain fatty acids, long-chain fatty esters, long-chainfatty alcohols, etc.), and which is a variant of a precursorthioesterase that comprises an analogous sequence to SEQ ID NO:31 inFIG. 58, wherein the precursor thioesterase is mutated with one or moresubstitution selected from: A1C, A1F, A1L, A1Y, D2L, D2M, D2P, D2W, T3R,L4A, L4M, L4N, L4S, L4V, L4Y, L5E, L5F, L5G, L5K, L5N, L5S, L5W, I6T,L7A, L7E, L7K, L7M, L7W, G8K, D9N, D9T, L11A, L11C, L11I, L11M, L11Q,L11V, S12I, S12L, S12M, S12N, S12T, S12V, S12Y, A13C, G14C, G14E, G14I,G14M, G14N, G14P, G14S, G14T, G14V, Y15C, Y15E, Y15G, Y15I, Y15N, Y15V,R16T, M17D, M17E, M17G, M17L, M17N, M17P, M17R, M17S, M17V, S18M, S18N,S18T, A19E, A19L, A19V, A21P, A22D, A22E, A22F, A22H, A22I, A22K, A22L,A22P, A22R, A22S, A22T, A22Y, W23A, W23H, W23N, W23P, P24A, P24C, P24D,P24E, P24F, P24G, P24I, P24M, P24N, P24S, P24T, P24V, P24W, L26P, L27A,L27C, L27F, L27H, L27R, L27S, L27T, L27W, L27Y, K30P, W31D, W31P, W31R,S36F, S36L, V37G, V37H, V37N, V37Q, V37W, V37Y, V38P, N39E, N39G, N39K,N39M, N39P, N39Q, N39Y, I42D, I42G, I42P, G44A, G44E, G44K, G44M, G44N,G44R, G44S, G44W, G44Y, D45G, D45M, T46D, S47E, S47P, S47Q, S47R, S47Y,Q48Y, G50C, G50E, G50F, G50I, G50K, G50L, G50M, G50N, G50P, G50Q, G50R,G50S, G50T, G50W, G50Y, L51D, L51P, L51T, A52P, R53A, R53C, R53D, R53E,R53F, R53G, R53I, R53K, R53L, R53N, R53S, R53T, R53V, R53W, R53Y, L54C,L54E, L54G, L54N, L54Y, P55Y, L57P, H61A, H61D, H61E, P63D, P63E, P63G,P63K, P63M, P63N, P63Q, P63R, R64L, W65G, W65P, W65R, V66N, V66Q, V66S,V66W, V66Y, L67E, L67G, L67Q, L67R, L67S, L67W, V68E, V68G, V68N, V68P,V68Q, E69A, E69C, E69D, E69F, E69G, E69H, E69K, E69L, E69M, E69N, E69P,E69Q, E69S, E69V, E69W, E69Y, L70A, L70C, L70E, L70F, L70G, L70H, L70K,L70Q, L70S, L70T, L70W, G71C, G71S, G72A, G72M, G72P, N73A, N73G, N73H,N73I, N73L, N73P, N73R, N73S, N73T, N73W, D74A, D74C, D74F, D74G, D74Q,D74S, D74W, D74Y, G75A, G75C, G75D, G75E, G75F, G75I, G75K, G75L, G75M,G75N, G75P, G75R, G75T, G75V, G75W, G75Y, L76A, L76C, L76D, L76E, L76F,L76G, L76I, L76K, L76M, L76N, L76P, L76Q, L76R, L76T, L76V, L76W, R77A,R77C, R77D, R77E, R77F, R77G, R77H, R77N, R77S, R77V, R77W, G78A, G78C,G78D, G78E, G78N, G78P, G78Q, G78Y, F79P, F79Q, F79S, F79V, P81E, P81W,T84D, T84E, T84G, T84H, T84K, T84L, T84N, T84Q, T84R, T84W, T84Y, E85F,E85P, Q86A, T87F, L88A, L88E, L88G, L88H, L88Q, L88S, L88W, L88Y, R89P,Q90P, Q90W, I91E, I91L, I91M, I91N, I91Q, I91S, I91Y, L92C, L92E, L92G,L92H, L92N, L92Q, L92R, L92S, L92Y, Q93P, D94P, D94V, V95A, V95C, V95D,V95E, V95F, V95I, V95P, V95Q, V95W, V95Y, K96P, A97C, A97P, N99D, A100Q,A100Y, P102E, P102G, P102H, P102L, P102R, P102V, P102W, L103C, L103E,L103I, L103K, L103N, L103R, L103S, L103T, L103V, L104A, L104C, L104E,L104G, L104I, L104N, L104P, L104Q, L104S, L104W, L104Y, M105A, M105C,M105E, M105F, M105G, M105K, M105L, M105P, M105T, M105W, Q106D, Q106G,Q106H, Q106L, Q106W, I107A, 1107E, I107F, I107G, I107K, I107L, I107Q,I107S, I107T, I107Y, R108A, R108C, R108D, R108E, R108F, R108G, R108H,R108I, R108L, R108M, R108S, R108V, R108Y, L109C, L109F, L109G, L109K,L109Q, L109R, L109T, L109V, L109Y, P110A, P110C, P110D, P110E, P110F,P110G, P110H, P110K, P110L, P110M, P110N, P110R, P110S, P110V, P110W,AMC, A111L, A111P, A111Q, A111R, A111V, N112I, N112L, N112P, N112Y,Y113D, Y113E, Y113Q, G114A, R115W, Y117D, Y117G, Y117P, N118F, E119C,E119L, A120P, F121A, F121C, F121D, F121E, F121G, F121K, F121L, F121N,F121P, F121Q, F121R, F121S, F121V, F121W, F121Y, S122D, S122E, S122L,S122P, I124D, I124E, I124G, I124H, I124K, I124R, I124W, I124Y, Y125C,Y125G, Y125H, Y125I, Y125L, Y125P, Y125Q, Y125R, Y125S, Y125T, Y125V,K127A, L128E, L128F, L128G, L128K, L128Q, L128R, L128S, L128W, A129D,A129F, A129L, A129W, A129Y, K130P, K130V, E131A, E131C, E131D, E131P,E131V, F132P, D133C, V134C, V134D, V134N, V134P, V134W, L136A, L136D,L136E, L136G, L136N, L136P, L136T, L137D, L137E, L137G, L137H, L137K,L137P, L137Q, L137R, L137S, P138G, P138N, P138V, F139A, F139C, F139D,F139E, F139G, F139H, F139M, F139N, F139S, F139T, F139V, F139W, F140A,F140C, F140G, F140I, F140L, F140M, F140N, F140P, F140S, F140T, F140V,F140W, M141C, M141D, M141E, M141F, M141G, M141K, M141L, M141P, M141Q,M141R, M141T, M141W, M141Y, E142A, E142C, E142G, E142I, E142L, E142M,E142P, E142Q, E142R, E142T, E142V, E143A, E143D, E143F, E143G, E143I,E143M, E143P, E143W, V144A, V144D, V144E, V144G, V144H, V144N, V144P,V144Q, V144R, V144S, Y145Q, Y145W, L146C, L146P, W150P, W150R, M151A,M151C, M151D, M151E, M151F, M151G, M151I, M151L, M151Q, M151R, M151S,M151T, M151V, M151W, Q152P, D153A, D153E, D153F, D154A, D154C, D154E,D154F, D154G, D154H, D154I, D154K, D154L, D154M, D154N, D154P, D154R,D154S, D154T, D154V, D154W, G155A, G155P, G155V, I156A, I156C, I156E,I156F, I156G, I156K, I156M, I156Q, I156R, I156S, I156T, I156Y, H157C,H157E, P158F, P158H, P158I, P158L, P158Q, P158V, P158W, N159P, N159W,A162K, A162L, A162N, A162R, A162Y, Q163A, Q163D, Q163E, Q163F, Q163I,Q163V, Q163W, Q163Y, F165L, I166A, I166F, I166M, I166S, I166Y, A167C,A167D, A167E, A167F, A167L, A167N, A167R, A167V, A167W, A167Y, D168M,D168R, M170E, M170F, M170G, M170N, M170S, M170T, A171S, Q173D, Q173P,L174A, L174G, L174S, L174T, L174W, L174Y, Q175F, P176L, P176Y, L177F,L177M, L177S, D181C, D181E, and/or D181G, wherein the numbers in thesubstitution mutation designations refer to amino acid positions of SEQID NO:31. An exemplary short-chain fatty acid derivative is a C₁₂ fattyacid derivative. An alternative short-chain fatty acid derivative is aC₁₄ fatty acid derivative. In certain circumstances, the decreasedproportional or percentage yield of short-chain fatty acid derivativescan be correlated to an increased proportional yield of long-chain fattyacid derivatives. The decreased proportional or percentage yield ofshort-chain fatty acid derivatives and/or the corresponding increasedproportional yield of long-chain fatty acid derivatives can be measuredin vitro or in vivo. Preferably, the decreased proportional yield ofshort-chain fatty acid derivatives or the corresponding increasedproportional yield of short-chain fatty acid derivatives is measured invivo.

In one embodiment of the invention, a polynucleotide (or a gene)encoding a mutant thioesterase (or a naturally-occurring equivalentthereof) of the invention is provided. In another embodiment of theinvention, a vector is provided comprising the polynucleotide (or thegene) according to the invention.

In one embodiment of the invention, the precursor thioesterase isencoded by a gene that is selectively hybridizable to the polynucleotidesequence of 'tesA, or an ortholog, paralog or homolog thereof. FIG. 56lists GenBank Accession Numbers of protein homologs of 'TesA having atleast 40% amino acid sequence identity to 'TesA. The precursorthioesterase can be encoded by a polynucleotide that is selectivelyhybridizable under conditions of intermediate stringency, under highstringency, or under maximum stringency.

In one embodiment of the invention, a polynucleotide encoding aprecursor thioesterase is provided wherein the precursor thioesterasecomprises the amino acid sequence of 'TesA, an ortholog thereof, aparalog thereof, or a homolog thereof. For example, the precursorthioesterase comprises the amino acid sequence of a 'TesA obtained froman E. coli, such as an E. coli K12. In a particular embodiment, apolynucleotide encoding the precursor thioesterase is provided whereinthe precursor thioesterase comprises the amino acid sequence, a variant,or a fragment of SEQ ID NO:31 of FIG. 58. In a particular embodiment,the gene encoding the precursor thioesterase comprises thepolynucleotide sequence of SEQ ID NO:32 in FIG. 59, or a fragmentthereof.

In one embodiment of the invention, a polynucleotide encoding aprecursor thioesterase is provided wherein the precursor thioesterasecomprises a protein having at least about 20%, for example, at leastabout 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity tothe sequence SEQ ID NO:31 of FIG. 58. In one embodiment, apolynucleotide encoding a precursor thioesterase is provided wherein theprecursor thioesterase comprises a protein having at least about 20%,for example, at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to the sequence of an E. coli K12 'TesA. In one embodimentof the invention, a polynucleotide is provided, which comprises asequence having at least about 20%, for example, at least about 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:32in FIG. 59.

In one embodiment of the invention, a vector is provided that comprisesa gene (or a polynucleotide) encoding a mutant thioesterase or anaturally-occurring equivalent thereof. Vectors according to theinvention can be transformed into suitable host cells to producerecombinant host cells.

In one embodiment of the invention, a probe is provided that comprises apolynucleotide of about 4 to about 150 nucleotides long, which issubstantially identical to a corresponding fragment of SEQ ID NO:32 inFIG. 59, wherein the probe is useful for detecting and/or identifyingpolynucleotide sequences encoding enzymes that have thioesteraseactivity. A probe according to the invention can be used to detect andisolate potential precursor thioesterases from sources not known toproduce such precursor thioesterases or for which the amino acid ornucleic sequence is unknown.

In certain embodiments of the invention, a recombinant host cell isprovided comprising a polynucleotide encoding a mutant thioesterase or anaturally-occurring equivalent thereof. In one embodiment, known genomicalteration or modification techniques can be employed to alter or modifythe endogenous thioesterases of the host cell, effectuating one or moreof the aforementioned mutations, such that at least one of the mutantendogenous thioesterases has at least one altered property. In anotherembodiment, the recombinant host cell is engineered to include a plasmidcomprising a polynucleotide encoding a mutant thioesterase or anaturally-occurring equivalent thereof. In yet another embodiment, therecombinant host cell expresses the thioesterase after thepolynucleotide encoding the thioesterase is integrated into thechromosome of the host cell.

In one embodiment of the invention, the recombinant host cell of theinvention can be selected from any cell capable of expressing arecombinant gene construct, and can be selected from a microbial, plantor animal cell. In a particular embodiment, the host cell is bacterial,cyanobacterial, fungal, yeast, algal, human or mammalian in origin. In aparticular embodiment, the host cell is selected from any of Grampositive bacterial species such as Actinomycetes; Bacillaceae, includingBacillus alkalophilus, Bacillus subtilis, Bacillus licheniformis,Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacilluscirculans, Bacillus lautus, Bacillus megaterium, B. thuringiensis;Brevibacteria sp., including Brevibacterium flavum, Brevibacteriumlactofermentum, Brevibacterium ammoniagenes, Brevibacterium butanicum,Brevibacterium divaricatum, Brevibacterium healii, Brevibacteriumketoglutamicum, Brevibacterium ketosoreductum, Brevibacteriumlactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum;Corynebacterium spp. such as C. glutamicum and C. melassecola,Corynebacterium herculis, Corynebacterium lilium, Corynebactertiumacetoacidophilum, Corynebacterium acetoglutamicum, Corynebacteriumacetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense,Corynebacterium nitrilophilus; or lactic acid bacterial speciesincluding Lactococcus spp. such as Lactococcus lactic; Lactobacillusspp. including Lactobacillus reuteri; Leuconostoc spp.; Pediococcusspp.; Serratia spp. such as Serratia marcescens; Streptomyces species,such as Streptomyces lividans, Streptomyces murinus, S. coelicolor andStreptococcus spp. Alternatively, strains of a Gram negative bacterialspecies belonging to Enterobacteriaceae including E. coli, Cellulomonasspp.; or to Pseudomonadaceae including Pseudomonas aeruginosa,Pseudomonas alcaligenes, Pseudomonas fluorescens, Pseudomonas putida,Pseudomonas syringae and Burkholderia cepacia, Salmonella sp.,Stenotrophomonas spp., and Stenotrophomonas maltophilia. Oleaginousmicroorganisms such as Rhodococcus spp, Rhodococcus opacus, Ralstoniaspp., and Acetinobacter spp. are useful as well. Furthermore, yeasts andfilamentous fungal strains can be useful host cells, including Absidiaspp.; Acremonium spp.; Agaricus spp.; Anaeromyces spp.; Aspergillusspp., including A. aculeatus, A. awamori, A. flavus, A. foetidus, A.fumaricus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreus;A. tubingensis and A. versicolor; Aeurobasidium spp.; Cephalosporumspp.; Chaetomium spp.; Coprinus spp.; Dactyllum spp.; Fusarium spp.,including F. conglomerans, F. decemcellulare, F. javanicum, F. lini, F.oxysporum and F. solani; Gliocladium spp.; Kluyveromyces sp.; Hansenulasp.; Humicola spp., including H. insolens and H. lanuginosa; Hypocreaspp.; Mucor spp.; Neurospora spp., including N. crassa and N. sitophila;Neocallimastix spp.; Orpinomyces spp.; Penicillium spp.; Phanerochaetespp.; Phlebia spp.; Pichia sp.; Piromyces spp.; Rhizopus spp.;Rhizomucor species such as Rhizomucor miehei; Schizophyllum spp.;Schizosaccharomyces such as, for example, S. pombe species; chytalidiumsp., Sulpholobus sp., Thermoplasma sp., Thermomyces sp.; Trametes spp.;Trichoderma spp., including T. reesei, T. reesei (longibrachiatum) andT. viride; Yarrowinia sp.; and Zygorhynchus spp and in particularinclude oleaginous yeast just Phafia spp., Rhorosporidium toruloides Y4,Rhodotorula Glutinis and Candida 107.

In one embodiment of the invention, a recombinant host cell is provided,which expresses or overexpresses a gene encoding the mutant thioesterase(or a naturally-occurring equivalent thereof), and which also expresses(or overexpresses) one or more genes encoding one or more enzymes thatutilize, as a substrates, reaction products of the mutant thioesterase(e.g., fatty acids, fatty acyl-COAs, fatty acyl-phosphate esters, fattyaldehydes, fatty esters, or fatty alcohols) or reaction products of oneor more other enzymes that are parts of a metabolic pathway, includingreaction products of the mutant thioesterase (e.g., fatty acids) asprecursors and/or substrates.

In one embodiment of the invention, a recombinant host cell is provided,which expresses or overexpresses a gene encoding a mutant thioesterase(or a naturally-occurring equivalent thereof) and which also expresses(or overexpresses) one or more genes encoding one or more enzymes thatreact with a substrate that is necessary as a precursor to a reaction ina fatty acid biosynthetic pathway. In a particular embodiment, therecombinant host cell includes a gene that encodes thioesterase and agene that encodes an enzyme that reacts with a substrate that isnecessary as a precursor to a reaction in a fatty acid syntheticpathway, which comprises the overexpression or modification of a geneselected from pdh, panK, aceEF, fabH, fabD, fabG, acpP, and/or fabF.

In one embodiment of the invention, the recombinant host cell comprisesa gene (or a polynucleotide) that encodes a mutant thioesterase (or anaturally-occurring equivalent thereof) and also comprises theattenuation or deletion of a gene that reduces carbon flowthrough, or agene that competes for substrates, cofactors, or energy requirementswithin a fatty acid biosynthetic pathway. In a particular embodiment,the attenuated gene comprises at least one of fadE, gpsA, ldhA, pflB,adhE, pta, poxB, ackA, ackB, plsB, and/or sfa.

In one embodiment of the invention, a recombinant host cell comprises agene (or a polynucleotide) encoding a mutant thioesterase (or anaturally-occurring equivalent thereof) and a heterologously-introducedexogenous gene encoding at least one fatty acid derivative enzyme. Incertain embodiments, the exogenous gene or polynucleotide encodes, forexample, an acyl-CoA synthase, an ester synthase, an alcoholacyltransferase, an alcohol dehydrogenase, an acyl-CoA reductase, afatty-alcohol-forming acyl-CoA reductase, a carboxylic acid reductase, adecarboxylase, an aldehyde reductase, a fatty alcohol acetyltransferase, an acyl condensing enzyme, an aminotransferase, or adecarbonylase.

In one embodiment of the invention, the recombinant host cell comprisesa gene encoding a mutant thioesterase (or a naturally-occurringequivalent thereof) and at least two heterologously-introduced exogenousgenes encoding fatty acid derivative enzymes. In certain embodiments,the exogenous genes or polynucleotides encode, for example, an acyl-CoAsynthase, an ester synthase, an alcohol acyltransferase, an alcoholdehydrogenase, an acyl-CoA reductase, a fatty-alcohol-forming acyl-CoAreductase, a carboxylic acid reductase, a decarboxylase, an aldehydereductase, a fatty alcohol acetyl transferase, an acyl condensingenzyme, an aminotransferase, or a decarbonylase.

In a preferred embodiment of the invention, a gene encoding the mutantthioesterase (or a naturally-occurring equivalent thereof) and/or afatty acid derivative enzyme, for example, an acyl-CoA synthase, anester synthase, an alcohol acyltransferase, an alcohol dehydrogenase, anacyl-CoA reductase, a fatty-alcohol forming acyl-CoA reductase, acarboxylic acid reductase, a decarboxylase, an aldehyde reductase, afatty alcohol acetyl transferase, an acyl condensing enzyme, an alcoholacetyltransferase, an aminotransferase, an additional thioesterase or adecarbonylase that is overexpressed.

In one embodiment of the invention, genes encoding mutant thioesterases(or naturally-occurring equivalents thereof), fatty acid derivativeenzymes and/or other recombinantly expressed genes in a recombinant hostcell are modified to optimize at least one codon for expression in therecombinant host cell.

In one embodiment of the invention, the recombinant host cell comprisesat least one gene encoding a mutant thioesterase (or anaturally-occurring equivalent thereof) and a gene encoding an acyl-CoAsynthase. The acyl-CoA synthase can be any of fadD, fadK, BH3103, yhfL,pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p, orthe gene encoding the protein ZP_01644857. Other examples of acyl-CoAsynthase genes include fadDD35 from M. tuberculosis HR7Rv [NP_217021],yhfL from B. subtilis [NP_388908], fadD1 from P. aeruginosa PAO1[NP_251989], the gene encoding the protein ZP_01644857 fromStenotrophomonas maltophilia R551-3, or faa3p from Saccharomycescerevisiae [NP_012257].

In one embodiment of the invention, a recombinant host cell is providedcomprising at least one gene or polynucleotide encoding a mutantthioesterase (or a naturally-occurring equivalent thereof) and a gene orpolynucleotide encoding an ester synthase, such as an ester synthasegene obtained from Acinetobacter spp., Alcanivorax borkumensis,Arabidopsis thaliana, Saccharomyces cerevisiae, Homo sapiens, Simmondsiachinensis, Mortierella alpina, Cryptococcus curvatus, Alcanivoraxjadensis, Alcanivorax borkumensis, Acinetobacter sp. HO1-N, orRhodococcus opacus. Examples of ester synthase genes include wax/dgat,encoding a bifunctional ester synthase/acyl-CoA: diacylglycerolacyltransferase from Simmondsia chinensis, Acinetobacter sp. strainADP1, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacterjadensis, Arabidopsis thaliana, or Alkaligenes eutrophus. In a preferredembodiment, the gene encoding the ester synthase is overexpressed.

In one embodiment of the invention, the recombinant host cell comprisesat least one gene encoding a fatty aldehyde biosynthetic enzyme. A fattyaldehyde biosynthetic gene can be, for example, a carboxylic acidreductase gene (e.g., a car gene), having a polynucleotide sequenceand/or polypeptide motif listed in FIGS. 32 and 33, or a variantthereof. In some instances, the fatty aldehyde biosynthetic gene encodesone or more of the amino acid motifs depicted in FIG. 33.

In one embodiment of the invention, the recombinant host cell comprisesat least one fatty alcohol production gene. Fatty alcohol productiongenes include, for example, acr1. Fatty alcohol production genes aredescribed in, for example, PCT Publication Nos. 2008/119082 and2007/136762, the disclosures of which are herein incorporated byreference.

In one embodiment of the invention, the recombinant host cell comprisesa gene encoding a mutant thioesterase (or a naturally-occurringequivalent thereof) and a gene encoding at least one olefin producinggene. The gene may be a terminal olefin producing gene or an internalolefin producing gene. As examples of terminal olefin producing genes,those described in PCT Publication No. 2009/085278, including orf880,are appropriate. As examples of internal olefin producing genes, thosedescribed in PCT Publication No. 2008/147781 A2 are appropriate. Thedisclosures of PCT Publication Nos. 2009/085278 and 2008/147781 A2 areherein incorporated by reference.

In one embodiment of the invention, a recombinant host cell is providedcomprising at least one gene or polynucleotide encoding a mutantthioesterase (or a naturally-occurring equivalent thereof), and at leastone of (a) a gene or polynucleotide encoding a fatty acid derivativeenzyme and (b) a gene or polynucleotide encoding an acyl-CoAdehydrogenase enzyme that is attenuated. Preferably that gene encoding afatty acid derivative enzyme that is attenuated or deleted is endogenousto the host cell, encoding, for example, an acyl-CoA synthase, an estersynthase, an alcohol acyltransferase, an alcohol dehydrogenase, anacyl-CoA reductase, a carboxylic acid reductase, a decarbonylase, afatty alcohol acetyl transferase, a fatty acid decarboxylase, or afatty-alcohol-forming acyl-CoA reductase. In one embodiment, theattenuated gene encodes an acyl-CoA synthase or an ester synthase.

In one embodiment of the invention, a recombinant host cell is providedthat expresses, or preferably overexpresses, a thioesterase enzyme underconditions that result in the direct synthesis of fatty esters fromacyl-ACP or acyl-CoA, such as fatty acid methyl esters (FAME) and fattyacid ethyl esters (FAEE), by such thioesterase. In this embodiment, thethioesterase directly converts acyl-ACP or acyl-CoA to fatty esterwithout necessarily expressing an enzyme that is a fatty acyl CoAsynthase or an ester synthase to produce fatty esters. Nonetheless,while expression or overexpression of a fatty acyl-CoA synthase or estersynthase is unnecessary, such enzymes may be desirable to increaseproduct yields. In this embodiment, the thioesterase enzyme can be anyof an endogenous thioesterase, a heterologously-expressed thioesterase,a mutant thioesterase, or a naturally-occurring equivalent thereof.

In one embodiment of the invention, the recombinant host cell has anendogenous gene encoding an acyl-CoA dehydrogenase enzyme that isdeleted or attenuated.

In one embodiment of the invention, a method is provided wherein therecombinant host cell according to the invention is cultured underconditions that permit expression or overexpression of one or morethioesterase enzymes, which can be selected from endogenousthioesterases, heterologously-expressed thioesterases, mutantthioesterases (or naturally-occurring equivalents thereof), or acombination of these thioesterases. In a particular embodiment, thethioesterase enzyme that is expressed or overexpressed can be recovered,and more preferably substantially purified, after the host cell isharvested and/or lysed.

In one embodiment of the invention, a method is provided wherein therecombinant host cell according to the invention is cultivated underconditions that permit production of fatty acid derivatives. In apreferred embodiment, the fatty acid derivative can be recovered, andmore preferably the fatty acid derivative is substantially purified. Ina particularly preferred embodiment, the fatty acid derivativecomposition is substantially purified from other components producedduring cultivation by centrifugation.

In one aspect of the invention, a method is provided for producing afatty acid derivative, comprising cultivating a recombinant host cell ofthe invention under conditions suitable to ensure expression oroverexpression of a mutant thioesterase (or a naturally-occurringequivalent thereof), and recovering the fatty acid derivative that isproduced.

In one embodiment, a method is provided for extracellularly producing afatty acid derivative in vitro, comprising cultivating a recombinanthost cell under conditions suitable for expression or overexpression ofa thioesterase enzyme (including, for example, an endogenousthioesterase, a heterologously-expressed thioesterase, a mutantthioesterase, or a naturally-occurring equivalent thereof), harvestingthe cells, and lysing the cells, such that the thioesterase enzyme thatis produced can be recovered and used to produce fatty acid derivativesin vitro. In an exemplary embodiment, the thioesterase enzyme issubstantially purified. In another exemplary embodiment, thethioesterase enzyme is not purified from the cell lysate. The purifiedthioesterase enzyme or the cell lysate comprising such an enzyme canthen be subject to suitable thioesterase substrates under conditionsthat allow the production of fatty acid derivatives extracellularly.Techniques for introducing substrates to enzymes are well known in theart. A non-limiting example is adding the substrate(s) in a solutionform to the enzyme solution or the cell lysate, and allowing the mixtureto incubate. Another non-limiting example involves incubating thesubstrate(s) and enzyme solution or cell lysate by either attaching thesubstrate(s) or the enzyme to a solid medium (e.g., beads, resins,plates, etc.) and pass the enzyme solution/lysate or the substrate(s),respectively through the solid medium in a speed that allows forsufficient contact between the substrate(s) and the enzyme.

In another embodiment of the invention, a method is provided forproducing a fatty acid derivative, which comprises cultivating arecombinant host cell under conditions suitable to ensure expression ofa thioesterase enzyme (including, for example, an endogenousthioesterase, a heterologously-expressed thioesterase, a mutantthioesterase, or a naturally-occurring equivalent thereof), andrecovering the fatty acid derivative that is secreted or releasedextracellularly. Accordingly, the fatty acid derivative product isrecovered from, for example, the supernatant of a fermentation brothwherein the host cell is cultured.

In one embodiment of the invention, a method is provided for obtaining afatty acid derivative composition extracellularly by cultivating arecombinant host cell that has been transformed with a polynucleotideencoding a thioesterase enzyme (including, for example, an endogenousthioesterase, a heterologous thioesterase, a mutant thioesterase, or anaturally-occurring equivalent thereof), cultivating under conditionsthat permit production of a fatty acid derivative, a major or minorportion of which is secreted or released extracellularly, and recoveringthe fatty acid derivative that is produced. In an exemplary embodiment,the fatty acid derivative is produced within the cell, but a portion ofit is released by the host cell. Accordingly, the method furthercomprises harvesting the cells, lysing the cells, and recovering thefatty acid derivative.

In one embodiment of the invention, a method of producing fatty acidderivatives is provided wherein a recombinant host cell that expresses,or preferably overexpresses, a thioesterase enzyme under conditions thatresult in the synthesis of fatty esters from acyl-ACP or acyl-CoA bysuch thioesterase is cultured under conditions that permit such directproduction of fatty esters.

In one embodiment of the invention, a method of producing fatty acidderivatives is provided comprising: modifying one or more endogenousthioesterases of the host cell using suitable genomic alterationtechniques such that the endogenous thioesterases comprise one or moremutations and have one or more altered properties, as compared to theendogenous thioesterase precursors; and cultivating the host cell underconditions suitable for said host cell to express or overexpress suchmutant thioesterases; and recovering the fatty acid derivatives. In anexemplary embodiment, the fatty acid derivative that is produced can besecreted or released extracellularly, such that it can be recoveredfrom, for example, the supernatant of the fermentation broth wherein thehost cell is cultured.

In one embodiment of the invention, a method of producing fatty acidderivatives is provided comprising: transforming the host cell with apolynucleotide sequence encoding a mutant thioesterase (or anaturally-occurring equivalent thereof), such that the production offatty acid derivatives in the host cell is altered relative to a cellthat has not been transformed with the mutant thioesterase gene (or anaturally-occurring equivalent thereof).

In one embodiment of the invention, a method of producing fatty acidderivatives is provided comprising: providing a polynucleotide sequencecomprising a gene encoding a mutant thioesterase (or anaturally-occurring equivalent thereof); transforming a suitable hostcell under conditions wherein said polynucleotide sequence isincorporated into said chromosome of said cell and said gene isexpressible within said host cell; cultivating the transformed host cellunder conditions suitable for said host cell to express said gene andproduce a mutant thioesterase protein (or a naturally-occurringequivalent thereof); and recovering the fatty acid derivatives.

In any of the embodiments above, derivatives of a certain carbon chainlength can be recovered at a greater proportional yield, in comparisonwith the production of such fatty acid derivatives of the same carbonchain length in the same host cell in the absence of the mutantthioesterase (or a naturally-occurring equivalent thereof). In aparticular embodiment, the fatty acid derivatives that are recovered atan increased or decreased yield comprise a primary chain length of C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁,C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆,C₃₇, C₃₈ or C₃₉ fatty acyl chain. The fatty acid derivatives that arerecovered at an increased or decreased yield in the composition can beselected from all types of fatty acid derivatives, including, forexample, hydrocarbons, fatty acids, fatty esters, fatty aldehydes, fattyalcohols terminal olefins, internal olefins, alkanes, diols, fattyamines, dicarboxylic acids, or ketones, or combinations thereof.

Alternatively, in any of the embodiments above, a particular fatty acidderivative can be produced at an increased or decreased proportional orpercentage yield relative to the other fatty acid derivatives, whencompared to the proportional or percentage yield of that particularfatty acid derivative in the same host cell in the absence of the mutantthioesterase (or a naturally-occurring equivalent thereof). In aparticular embodiment, the fatty acid derivative that is produced at anincreased proportional or percentage yield is a fatty ester. In anotherembodiment, the fatty acid derivative that is produced at a decreasedproportional or percentage yield is a fatty ester.

Alternatively, in any of the embodiments above, fatty acid derivativescan be produced at an increased yield, or at an increased proportionalyield of short-chain (e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, or C₁₄)products. Conversely, in any of the embodiments above, fatty acidderivatives can be produced at a decreased yield, or at a decreasedproportional yield of short-chain (e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, orC₁₄) products.

In one embodiment of the invention, a method of producing fatty acidderivatives is provided wherein the yield of fatty acid derivativesproduced by the method of the invention is at least about 0.001 g offatty acid derivative product/g of carbon source, for example, at leastabout 0.01 g of fatty acid derivative product/g of carbon source, about0.1 g of fatty acid derivative product/g of carbon source, about 0.2 gof fatty acid derivative product/g of carbon source, about 0.3 g offatty acid derivative product/g of carbon source, about 0.4 g of fattyacid derivative product/g of carbon source, or about 0.45 g of fattyacid derivative product/g of carbon source.

In one embodiment of the invention, a method of producing fatty acidderivatives is provided wherein the method results in a titer of atleast about 0.5 g/L, for example, at least about 1 g/L, 2 g/L, 5 g/L, 10g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 75 g/L, 100 g/L, 150 g/L or 200g/L.

In one embodiment of the invention, a method of producing fatty acidderivatives is provided wherein the productivity of the method is suchthat at least about 0.1 g/L·h, for example, at least about 0.5 g/L·h, 1g/L·h, 2 g/L·h, 3 g/L·h, 4 g/L·h, 5 g/L·h, 6 g/L·h, 7 g/L·h or 8 g/L·his produced.

In one embodiment of the invention, fatty acid derivative compositionsare provided that are produced by the host cells of the invention. Suchcompositions can comprise hydrocarbons, esters, alcohols, ketones,aldehydes, fatty acids, dicarboxylic acids, internal olefins, terminalolefins, and/or combinations thereof. Such compositions are useful inapplications in the chemical industry, for example in the production ofsurfactants and detergents, or as a biofuel and a substitute forpetroleum, heating oil, kerosene, diesel, jet fuel or gasoline.

In one embodiment of the invention, fatty acid derivative compositionsare provided comprising less than or equal to about 50 ppm arsenic,about 30 ppm, about 25 ppm, or between about 10 and about 50 ppmarsenic; less than or equal to about 200 ppm calcium, about 150 ppmcalcium, about 119 ppm calcium or between about 50 and about 200 ppmcalcium; less than or equal to about 200 ppm chlorine, about 150 ppmchlorine, about 119 ppm chlorine or between about 50 and about 200 ppmchlorine; less than or equal to about 50 ppm copper, about 30 ppmcopper, about 23 ppm copper, or between about 10 and about 50 ppmcopper; less than or equal to about 300 ppm iron, about 200 ppm iron,about 136 ppm iron, or between about 50 and about 250 ppm iron; lessthan or equal to about 50 ppm lead, about 30 ppm lead, about 25 ppmlead, or between about 10 and about 50 ppm lead; less than or equal toabout 50 ppm manganese, about 30 ppm manganese, about 23 ppm manganese,or between about 10 and about 50 ppm manganese; less than or equal toabout 50 ppm magnesium, about 30 ppm magnesium, about 23 ppm magnesium,or between about 10 and about 50 ppm magnesium; less than or equal toabout 0.5 ppm mercury, about 0.1 ppm mercury, about 0.06 ppm mercury orbetween about 0.01 and about 0.2 ppm mercury; less than or equal toabout 50 ppm molybdenum, about 30 ppm molybdenum, about 23 ppmmolybdenum or between about 10 and about 50 ppm molybdenum; less than orequal to about 2% nitrogen; about 1% nitrogen, about 0.5% nitrogen, orbetween about 0.1-1% nitrogen; less than or equal to about 200 ppmpotassium, about 150 ppm potassium, about 103 ppm potassium, or betweenabout 50 and about 200 ppm potassium; less than or equal to about 300ppm sodium, 200 ppm sodium, about 140 ppm sodium, or between about 50and about 300 ppm sodium; less than or equal to about 1 ppm sulfur, lessthan or equal to about 1% sulfur, about 0.14% sulfur, or between about0.05 and about 0.3% sulfur; less than or equal to about 50 ppm zinc,about 30 ppm zinc, about 23 ppm zinc, or between about 10 and about 50ppm zinc; or less than or equal to about 700 ppm phosphorus, about 500ppm phosphorus, about 350 ppm phosphorus, or between about 100 and about700 ppm phosphorus.

In one embodiment of the invention, fatty acid derivatives havingfractions of modern carbon of about 1.003 to about 1.5 are provided.

In one embodiment of the invention, a fatty acid derivative compositionis provided wherein the composition includes constituents comprising anacyl group that has a double bond at position 7 in the carbon chain(between carbon number 7 on the carbon chain and carbon number 8 on thecarbon chain) from its reduced end.

In a particular embodiment, the fatty acid derivative compositioncomprises C₅-C₂₅ (i.e., a carbon chain length of 5 to 25 carbons) fattyesters, C₅-C₂₅ fatty acids, C₅-C₂₅ fatty aldehydes, C₅-C₂₅ fattyalcohols; or C₁₀-C₂₀ (i.e., a carbon chain length of 10 to 20 carbons)fatty esters, C₁₀-C₂₀ fatty acids, C₁₀-C₂₀ fatty aldehydes, C₁₀-C₂₀fatty alcohols; or C₁₂-C₁₅ (i.e., a carbon chain length of 12 to 18carbons) fatty esters, C₁₂-C₁₈ fatty acids, C₁₂-C₁₈ fatty aldehydes,C₁₂-C₁₈ fatty alcohols.

In a particular embodiment, the fatty acid derivatives of the inventioncomprise straight chain fatty acid derivatives, branched chain fattyacid derivatives, and/or cyclic moieties. In a particular embodiment,the fatty acid derivatives are unsaturated (e.g., monounsaturated) orsaturated.

In one embodiment of the invention, the fatty acid derivativecomposition comprises a fatty ester that is produced from an alcohol andan acyl-CoA, wherein the alcohol is at least about 1, for example, atleast about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 10, about 12, about 14, about 16, or about 18 carbons in length,and the acyl-CoA is at least about 2, for example, at least about 4,about 6, about 8, about 10, about 12, about 14, about 16, about 18,about 20, about 22, about 24, or about 26 carbons in length. In someembodiments, the alcohol and acyl-CoA from which the fatty ester areproduced vary by about 2, about 4, about 6, about 8, about 10, about 12,or about 14 carbon atoms.

In another embodiment, the fatty acid derivative composition comprises afatty ester that is produced from an alcohol and an acyl-ACP, whereinthe alcohol is at least about 1, for example, at least about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 10, about 12, about14, about 16, or about 18 carbons in length, and the acyl-ACP is atleast about 2, for example, about 4, about 6, about 8, about 10, about12, about 14, about 16, about 18, about 20, about 22, about 24, or about26 carbons in length. In some embodiments, the alcohol and acyl-ACP fromwhich the fatty ester are produced vary by about 2, about 4, about 6,about 8, about 10, about 12 or about 14 carbon atoms.

In one embodiment of the invention, the fatty acid derivativecomposition comprises a mixture of derivatives including free fattyacids. In one embodiment, the percentage of free fatty acids by weightis at least about 0.5%, for example, at least about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 15%, about 20%, or about 25%. In a certain embodiment, thepercentage of fatty esters produced by weight is at least about 50%, forexample, at least about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, or about 90%. In a further embodiment, the ratioof fatty acid derivatives other than free fatty acids to free fattyacids is greater than about 90:1, for example, greater than about 80:1,about 50:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1,about 5:1, about 2:1 or about 1:1, by weight.

In one embodiment, the fatty acid derivative composition comprises amixture of derivatives including free fatty acids. In one embodiment,the percentage of free fatty acids by weight is at least about 50%, forexample, at least about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, or about 90%. In a certain embodiment, thepercentage of fatty ester produced by weight is at least about at leastabout 0.5%, for example, at least about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,or about 50%. In a further embodiment, the ratio of the fatty acidderivative produced other than free fatty acids to free fatty acids isless than about 60:1, for example, less than about 50:1, about 40:1,about 30:1, about 20:1, about 10:1, about 1:1, about 1:2; about 1:3,about 1:5, or about 1:10, by weight.

In one embodiment of the invention, the fatty acid derivativecomposition includes one or more fatty esters selected from: ethyldecanoate, ethyl dodecanoate, ethyl tridecanoate, ethyl tetradecanoate,ethyl pentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate,ethyl heptadecanoate, ethyl cis-11-octadecenoate, ethyl octadecanoate,methyl decanoate, methyl dodecanoate, methyl tridecanoate, methyltetradecanoate, methyl pentadecanoate, methyl cis-9-hexadecenoate,methyl hexadecanoate, methyl heptadecanoate, methylcis-11-octadecenoate, methyl octadecanoate, or a combination thereof.

In one embodiment of the invention, the fatty acid derivativecomposition includes one or more free fatty acids selected from:octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid,pentadecanoic acid, cis-9-hexadecenoic acid, hexadecanoic acid,cis-11-octadecenoic acid, or combinations thereof.

Compositions comprising the fatty acid derivatives of the invention canbe used as fuels. For example, the fatty acid derivatives can be usedas, or as a component of, a biodiesel, a fatty alcohol, a fatty ester, atriacylglyceride, a gasoline, a diesel, or a jet fuel. A gasoline or abiodiesel composition can be used in an internal combustion engine. Ajet fuel can be used in a jet engine. Accordingly, fuel compositionscomprising the fatty acid derivatives prepared according to the presentdisclosures are provided herein.

Compositions comprising fatty acid derivatives of the invention can beused as fuel additives. For example, they can be added to apetroleum-based diesel or biodiesel to improve its renewable fuelcontent, lubricity, kinematic viscosity, acid number, boiling point,oxidative stability, cold filter-plugging point, impurity profiles,sulfated ash level, cetane number, cloud point, or pour point.Accordingly, fuel additive compositions comprising fatty acidderivatives produced according to the present disclosures are alsoprovided.

Compositions comprising fatty acid derivatives of the invention can alsobe used as biocrude compositions, which can serve as feedstocks formaking other petroleum-derivative compounds. For example, long chainhydrocarbons, internal or terminal olefins, alkanes, fatty aldehydes andfatty esters made according to the current invention can be furtherprocessed to produce fuels, fuel additives, fuel blends, and/or chemicalproducts. Accordingly, biocrude compositions comprising fatty acidderivatives prepared according to the present disclosures are provided.

Compositions comprising fatty acid derivatives of the invention can beused as feedstocks in manufacturing detergents and surfactants,nutritional supplements, polymers, paraffin replacements, lubricants,solvents, personal care products, rubber processing additives, corrosioninhibitors, emulsifiers, plastics, textiles, cosmetics, paper products,coatings, metalworking fluids, dielectrics, oiling agents, and/oremollients. Accordingly, feedstock compositions comprising fatty acidderivatives prepared according to the present disclosures are alsoprovided.

DESCRIPTION OF THE FIGURES

FIG. 1 is a table identifying various genes that can be over-expressedor attenuated to increase fatty acid derivative production. The tablealso identifies various genes that can be modulated to alter thestructure of the fatty acid derivative product. Certain of the genesthat are used to alter the structure of the fatty acid derivative willalso increase the production of fatty acid derivatives.

FIG. 2 is a diagram illustrating the beta-oxidation pathway, includingsteps catalyzed by the following enzymes (1) acyl-CoA synthase (EC6.2.1.−). (2) acyl-CoA dehydrogenase (EC 1.3.99.3), (3) enoyl-CoAhydratase (EC 4.2.1.17); (4) 3-hydroxybutyryl-CoA epimerase (EC5.1.2.3), and (5) 3-ketoacyl-CoA thiolase (EC 2.3.1.16). This finalreaction of the β-oxidation cycle, releases acetyl-CoA and an acyl-CoAfatty acid two carbons shorter, ready to go through β-oxidationreactions again.

FIG. 3 is a diagram illustrating the FAS biosynthetic pathway.

FIG. 4 is a diagram illustrating biosynthetic pathways that producefatty esters depending upon the substrates provided.

FIG. 5 is a diagram illustrating biosynthetic pathways that producefatty alcohols.

FIG. 6 is a graph depicting fatty alcohol production by the strainco-transformed with pCDFDuet-1-fadD-acr1 and plasmids containing variousthioesterase genes. Saturated C₁₀, C₁₂, C₁₄, C₁₆ and C₁₈ fatty alcoholwere identified.

FIG. 7 is a graph depicting fatty alcohol production by the straindescribed in Example 3, co-transformed with pCDFDuet-1-fadD-acr1 andplasmids containing various thioesterase genes. The strains were grownaerobically at 25° C. or 37° C. in an M9 mineral medium containing 0.4%glucose in shake flasks. Fatty alcohols were detected in the cellpellets as well as in the supernatants, indicating a substantialextracellular production of such alcohols. Cultivation at 25° C.resulted in the release of about 25% of the product from the cells,whereas cultivation at 37° C. resulted in the release of about 50% ofthe product from the cell.

FIG. 8A-D are plots depicting GC-MS spectra of octyl octanoate (C₈C₈)produced by a production host expressing alcohol acetyl transferase(AATs, EC 2.3.1.84) and production hosts expressing ester synthase (EC2.3.1.20, 2.3.1.75). FIG. 8A is a GC-MS spectrum showing ethyl acetateextract of strain C41(DE3, ΔfadE/pHZ1.43)/pRSET B+pAS004.114B) whereinthe pHZ1.43 plasmid expressed ADP1 ester synthase (EC 2.3.1.20,2.3.1.75). FIG. 8B is a GC-MS spectrum showing ethyl acetate extract ofstrain C41(DE3, ΔfadE/pHZ1.43)/pRSET B+pAS004.114B) wherein the pHZ1.43plasmid expressed SAAT. FIG. 8C is a GC-MS spectrum showing acetylacetate extract of strain C41(DE3, ΔfadE/pHZ1.43)/pRSET B+pAS004.114B)wherein the pHZ1.43 plasmid did not contain ADP1 (an ester synthase) orSAAT. FIG. 8D is a GC-MS spectrum showing the mass spectrum andfragmentation pattern of C₈C₈ produced by C41(DE3, ΔfadE/pHZ1.43)/pRSETB+pAS004.114B wherein the pHZ1.43 plasmid expressed SAAT).

FIG. 9 is a graph depicting the distribution of ethyl esters made (inaccordance with Example 9) when the ester synthase from A. baylyi ADP1(WSadp1) was co-expressed with a thioesterase from Cuphea hookeriana ina production host.

FIG. 10 is a graph depicting the production of ethyl esters by variousester synthases at 25° C. The ethyl esters were produced by recombinantE. coli strains carrying various ester synthase genes. The recombinantstrains were (1) C41 (DE3, ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadDwith 1 pHZ1.43; (2) pHZ1.97_377; (3) pHZ1.97_atfA2; (4) pHZ1.97_376; (5)pHZ1.97_atfA1; and (6) no plasmids (control).

FIG. 11 is a graph depicting the acyl composition of fatty acid ethylesters (FAEE) produced from various E. coli strains. The recombinantstrains are (1) C41 (DE3, ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadDwith 1 pHZ1.43; (2) pHZ1.97_377; (3) pHZ1.97_atfA2; (4) pHZ1.97_376; (5)pHZ1.97_atfA1; and (6) no plasmids (control).

FIG. 12 is a graph depicting the production of ethyl esters by variousester synthases at 37° C. The ethyl esters were produced by recombinantE. coli strains carrying various ester synthase genes. The recombinantstrains were (1) C41 (DE3, ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadDwith 1 pHZ1.43; (2) pHZ1.97_377; (3) pHZ1.97_atfA2; (4) pHZ1.97_376; (5)pHZ1.97_atfA1; and (6) no plasmids (control).

FIG. 13 is a graph depicting concentrations of free fatty acids (FFA)and fatty acid ethyl esters (FAEE) produced from three individualcolonies from the transformants, C41 (DE3,ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadD+pHZ1.97_atfA2. The FFA wasconverted to fatty acid ethyl ester (FAEE) and quantified by GC/MS.

FIG. 14 is a diagram depicting the control regions for FabA (SEQ IDNO:33) and FabB (SEQ ID NO:34). The FadR and FabR consensus bindingsites are shown in bold. Vertical arrows indicate the positions wheremutations can be made to alter fabA expression. The proposed base foreach position is also indicated by the brackets. The two regions thatconstitute the −35 and −10 regions of the typical E. coli promoter areindicated by the brackets. The proposed mutations that make the promotercloser to the consensus promoter sequence are also shown.

FIGS. 15A-B are chromatograms depicting GC/MS analysis. FIG. 15A is achromatogram depicting the components of an ethyl acetate extract of theculture of E. coli LS9001 strain transformed with plasmidspCDFDuet-1-fadD-WSadp1, pETDuet-1-'TesA. FIG. 15B is a chromatogramdepicting the ethyl hexadecanoate and the ethyl oleate, which were usedas references.

FIG. 16 is a map of the pOP-80 plasmid.

FIG. 17 is the full DNA sequence of the pOP-80 plasmid (SEQ ID NO:1)

FIG. 18 is the DNA sequence (SEQ ID NO:2) for the E. colicodon-optimized fadD35 gene (GenBank Accession No. NP_217021).

FIG. 19 is the DNA sequence (SEQ ID NO:3) for the E. colicodon-optimized fadD1 gene (GenBank Accession No. NP_251989).

FIG. 20 is the BsyhfLBspHIF primer (SEQ ID NO:4) based on the DNAsequence deposited at NCBI with GenBank Accession No. NC_000964.

FIG. 21 is the BsyhfLEcoR primer (SEQ ID NO:5) based on the DNA sequencedeposited at NCBI with GenBank Accession No. NC_000964.

FIG. 22 is the DNA sequence (SEQ ID NO:6) for the yhfL gene fromBacillus subtilis.

FIG. 23 is the Scfaa3pPciF primer (SEQ ID NO:7) based on the DNAsequence deposited at NCBI with GenBank Accession No. NC_001141.

FIG. 24 is the Scfaa3pPciI primer (SEQ ID NO:8) based on the DNAsequence deposited at NCBI with GenBank Accession No. NC_001141.

FIG. 25 is the DNA sequence (SEQ ID NO:9) for the faa3 gene fromSaccharomyces cerevisiae (GenBank Accession No. NP_012257).

FIG. 26 is the Smprk59BspF primer (SEQ ID NO:10) based on the DNAsequence deposited at NCBI with GenBank Accession No. NZ_AAVZ01000044.

FIG. 27 is the Smprk59HindR primer (SEQ ID NO:11) based on the DNAsequence deposited at NCBI with GenBank Accession No. NZ_AAVZ01000044.

FIG. 28 is the PrkBsp primer (SEQ ID NO:12).

FIG. 29 is the DNA sequence encoding the protein ZP_01644857 fromStenotrophomonas maltophilia R551-3 (SEQ ID NO:13).

FIG. 30 is the protein sequence of ZP_01644857 from Stenotrophomonasmaltophilia ATCC 17679 (SEQ ID NO:14).

FIG. 31 is a schematic of a new pathway for fatty aldehyde production.

FIG. 32 is a listing of the nucleotide sequence (SEQ ID NO:15) and thecorresponding amino acid sequence (SEQ ID NO:16) of Nocardia sp. NRRL5646 car gene.

FIG. 33 is a listing of amino acid sequence motifs for CAR homologs.

FIGS. 34A-B are GC/MS traces of olefins produced by Jeotgalicoccus sp.ATCC 8456 cells and Jeotgalicoccus halotolerans DSMZ 17274 cells,respectively.

FIGS. 35A-B are GC/MS traces of olefins produced by Jeotgalicoccuspinnipedalis DSMZ 17030 cells and Jeotgalicoccus psychrophilus DSMZ19085 cells, respectively.

FIGS. 36A-B are mass spectrometry fragmentation patterns of twoα-olefins produced by Jeotgalicoccus ATCC 8456 cells. Compound A wasidentified as 1-nonadecene and compound B as 18-methyl-1-nonadecene.

FIG. 37 is a schematic of a phylogenetic analysis of 16s rRNA ofJeotgalicoccus ATCC 8456.

FIGS. 38A-B are GC/MS traces of α-olefins produced by Jeotgalicoccus sp.ATCC 8456 cells upon feeding with eicosanoic acid (FIG. 38A) or stearicacid (FIG. 38B).

FIG. 39 is a GC/MS trace of α-olefins (1-heptadecene) produced by cellfree lysates of Jeotgalicoccus sp. ATCC 8456 cells, as compared to atrace of cell-free lysate without the Cis fatty acid substrate, and atrace of the Cis fatty acid substrate itself.

FIG. 40 is a digital representation of an SDS-PAGE gel of final purifiedα-olefins-producing protein fraction from Jeotgalicoccus sp. ATCC 8456cells.

FIGS. 41A-B are orf880 nucleotide (SEQ ID NO:25) and amino acid (SEQ IDNO:26) sequences, respectively. FIG. 41C is the partial 16s rRNAsequence (SEQ ID NO:27) of Jeotgalicoccus sp. ATCC8456.

FIG. 42 is a GC/MS trace of α-olefins produced by E. coli uponexpression of Jeotgalicoccus sp. 8456_orf880 and feeding of stearicacid.

FIG. 43 is a schematic of a bootstrap phylogenetic analysis of8456_orf880 homologs using ClustalW.

FIG. 44 describes amino acid motifs for identifying precursorthioesterases useful in the present invention.

FIGS. 45A-B include a tables listing the results of assays identifyingmutant thioesterases with altered properties. In particular, FIG. 45Aincludes lists of mutants with Z scores of at least 3 for activity(i.e., catalytic rate) with respect to the named substrate orspecificity for the named substrate; and FIG. 45B is a table of mutantshaving improved and/or increased yield/production of fatty acidderivatives with Z scores of at least 3.

FIGS. 46A-E include tables listing the results of assays identifyingmutant thioesterases with altered proportional yield of fatty esters vs.other products (e.g., fatty acid derivatives other than fatty esters).In particular, FIG. 46A is a table showing mutants having Z scores of atleast 3 with respect to the proportional or percentage yield of fattyesters vs. free fatty acids. FIG. 46B is a table showing mutants havingZ scores of less than −3 with respect to the proportional or percentageyield of fatty esters vs. free fatty acids. FIG. 46C is a table showingmutants having Z scores of at least 3 with respect to the in vivo yieldof fatty acid derivatives. FIG. 46D is a table showing mutants having Zscores of at least 3 with respect to the proportional yield ofshort-chain (e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, and/or C₁₄) fatty acidderivatives vs. other fatty acid derivatives (e.g., fatty acidderivatives other than short-chain fatty acid derivatives including, forexample, long-chain (e.g., C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, and/or C₂₀) fattyacid derivatives). FIG. 46E is a table showing mutants having Z scoresof less than −3 with respect to the proportional yield of short-chain(e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, and/or C₁₄) fatty acid derivativesvs. other fatty acid derivatives (e.g., fatty acid derivatives otherthan short-chain fatty acid derivatives including, for example,long-chain (e.g., C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, and/or C₂₀) fatty acidderivatives).

FIG. 47 is a sequence alignment of homologs of 'TesA using the aminoacid residues of an E. coli 'TesA (i.e., TesA without the signalpeptide) as a reference sequence for numbering purposes.

FIG. 48 is a graph depicting the FAME titers and composition for theMG1655 (ΔfadE) pTrc-'TesA_fadD strain.

FIG. 49 is a graph depicting the FAME titers and composition for theMG1655 (ΔfadE) and C41(ΔfadE) strains expressing fadD and 'tesA onplasmids during a 25-hour fermentation run.

FIG. 50 is a graph depicting the FAME titers and composition for theMG1655 (ΔfadE) pTrc-'TesA_fadD strain.

FIG. 51 is a graph depicting the FAME titers and composition for theMG1655 (ΔfadE) and C41 (ΔfadE) strains expressing fadD and 'tesA onplasmids during a 25-hour fermentation run.

FIG. 52 is a graph depicting the FFA titers and composition for theMG1655 (4fadE) and C41 (4fadE) strains expressing fadD and 'tesA onplasmids during a 25-hour fermentation run.

FIG. 53 is a graph depicting the FAME titers for the MG1655 (4fadE)strains expressing E. coli 'tesA, P. luminescens 'tesA, V. harveyi 'tesAand P. profundum tesB on plasmids, during a 24-hour fermentation run.Titers are represented in mg/L and mg/L/OD.

FIG. 54 is a graph of FFA titers for MG1655 (4fadE) strains expressingE. coli 'tesA, P. luminescens 'tesA, V. harveyi 'tesA and P. profundumtesB on plasmids, during a 24-hour fermentation run. Titers arerepresented in mg/L (bars) and mg/L/OD (triangles).

FIG. 55 compares the relevant sequence regions of naturally-occurringthioesterases that comprise residues at positions that correspond tomutations in 'TesA that introduce altered properties. The relevantresidue is highlighted in dark color, and aligned with correspondingresidues in naturally-occurring thioesterases.

FIG. 56 lists GenBank Accession numbers of 'TesA homologs.

FIGS. 57A-F are graphs depicting substrate specificity (Z score) vs.amino acid residue positions corresponding to 'TesA sequence of SEQ IDNO:31 with symbols to represent levels of conservation in the cons70alignment for C₁₀ specificity (FIG. 57A and FIG. 57D), C₁₂ specificity(FIG. 57B and FIG. 57E) and C₁₄ specificity (FIG. 57C and FIG. 57F).

FIG. 58 shows the amino acid sequence of an E. coli 'TesA (SEQ IDNO:31).

FIG. 59 shows a nucleotide sequence encoding an E. coli 'TesA (SEQ IDNO:32).

FIG. 60 is a graph of free fatty acid (FFA) and fatty acyl methyl ester(FAME) titers in cultures of E. coli MG1655 ΔfadE cells transformed withpACYC containing the 'tesA homologs from E. coli (EcolA), Pectobacteriumatrosepticum (PatrA), Pseudomonas putida (PputA), Vibrio harveyi(VharA), Photorhabdus luminescens (PlumA), or with pACYC containing noinsert (Neg).

FIG. 61 is a graph of FFA and FAME titers in cultures of E. coli MG1655ΔfadE cells overexpressing fadD and 'tesA from E. coli (Ecoli),Pectobacterium atrosepticum (Patr), Photorhabdus luminescens (Plum),Photobacterium profundum (Ppro), Vibrio harveyi (VhA), Pseudomonasputida (Pput), or no 'tesA (Neg). (Data marked with an asterisk (*) arefrom a separate experiment.)

FIG. 62 is a graph of FFA and FAME titers in cultures of E. coli MG1655ΔfadE expressing wildtype E. coli 'tesA (WT), the S10C mutant (S10C), orno 'tesA (Neg).

FIG. 63 is a graph of FAME production against time of a fermentation runwith recombinant host cells that express thioesterase in the absence ofexogenous ester synthase.

FIG. 64 is a graph of FFA production against time of a fermentation runwith recombinant host cells that express thioesterase in the absence ofexogenous ester synthase.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein, including GenBankdatabase sequences, are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

Definitions

Throughout the specification, a reference may be made using anabbreviation of a gene name or a polypeptide name, but it is understoodthat such an abbreviated gene or polypeptide name represents the genusof genes or polypeptides, respectively. Such gene names include allgenes encoding the same polypeptide and homologous polypeptides havingthe same physiological function. Polypeptide names include allpolypeptides that have the same activity (e.g., that catalyze the samefundamental chemical reaction).

Unless otherwise indicated, the accession numbers referenced herein arederived from the NCBI database (National Center for BiotechnologyInformation) maintained by the National Institute of Health, U.S.A.Unless otherwise indicated, the accession numbers are as provided in thedatabase as of March 2008.

EC numbers are established by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)(available at http://www.chem.qmul/ac/uk/iubmb/enzyme/). The EC numbersreferenced herein are derived from the KEGG Ligand database, maintainedby the Kyoto Encyclopedia of Genes and Genomics, sponsored in part bythe University of Tokyo. Unless otherwise indicated, the EC numbers areas provided in the database as of March 2008.

The articles “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

The term “about” is used herein to mean a value ±20% of a givennumerical value. Thus, “about 60%” refers to a value of 60±(20% of 60)(i.e., between 48 and 70).

As used herein, the term “alcohol dehydrogenase” (EC 1.1.1.*) is apolypeptide capable of catalyzing the conversion of a fatty aldehydes toan alcohol (e.g., a fatty alcohol). Additionally, one of ordinary skillin the art will appreciate that some alcohol dehydrogenases willcatalyze other reactions as well. For example, some alcoholdehydrogenases will accept other substrates in addition to fattyaldehydes. Such non-specific alcohol dehydrogenases are, therefore, alsoincluded in this definition. Polynucleotide sequences encoding alcoholdehydrogenases are known in the art, and such dehydrogenases arepublicly available.

The term “altered property” refers to a modification in one or moreproperties of a mutant polynucleotide or mutant protein with referenceto a precursor polynucleotide or precursor protein. Properties that canbe advantageously altered with respect to proteins made according to thepresent invention include oxidative stability, substrate specificity,substrate selectivity, catalytic activity, thermal stability, pHstability, pH activity profile, resistance to proteolytic degradation,K_(m), k_(cat), k_(cat)/k_(m) ratio, protein folding, inducing an immuneresponse, ability to bind to a ligand, ability to bind to a receptor,ability to be secreted, ability to translocate in an active manner intoa membrane, ability to be displayed on the surface of a cell, ability tooligomerize, ability to signal, ability to stimulate cell proliferation,ability to inhibit cell proliferation, ability to induce apoptosis,ability to be modified by phosphorylation or glycosylation, ability totreat disease. In one embodiment of the invention, mutant thioesterasesare provided that derive from a precursor thioesterase, wherein themutant has at least one altered property either in vitro or in vivo, ascompared to the properties of the precursor thioesterase. In oneembodiment, the altered property can be a biophysical property such asthermal stability (melting point T_(m)), solvent stability, solutestability, oxidative stability, lipophilicity, hydrophilicity,quaternary structure, dipole moment, or isoelectric point. In oneembodiment, the altered property can be a biochemical property such aspH optimum, temperature optimum, ionic strength optimum, and/or anenzyme catalytic parameter (such as, for example, product distribution,product proportional or percentage yield, specific activity, substratepreference, substrate affinity, substrate inhibition, product affinity,turnover rate, product inhibition, kinetic mechanism, K_(M), k_(cat),k_(cat)/K_(m), and/or V_(Max)). In one embodiment, the altered propertyis a changed preference for particular substrates, as reflected in, forexample, a changed preference for alcoholysis or hydrolysis, acyl-CoA oracyl-acyl carrier protein substrates, ester or thioester substrates,saturated or unsaturated substrates, position of unsaturations, broad ornarrow specificity (e.g., the ability to catalyze a range of substratesor only substrates of a specific carbon chain length). In oneembodiment, the altered property can be an increased preference oractivity for branched substrates, substrates having a specific positionof branching, hydroxy-acyl substrates, keto-acyl substrates, substratesthat result in a product having desirable fuel attributes (i.e., cetanenumber, octane rating, oxidative stability, lubricity, flash point,viscosity, boiling point, melting point, pour point, cloud point, coldfilter plugging point, cold flow characteristics, aromaticity, and/oriodine number). Altered properties also include a decrease in activityor attenuation of ester hydrolysis, such as hydrolysis of desiredproduct molecules, or a decrease in the toxicity of the protein to thecell and/or a change in the expression level of the protein in the cell.In a particular embodiment, the at least one altered property is, forexample, a change in the ability of the thioesterase to catalyze thesynthesis of fatty acyl esters directly or indirectly, in vivo or invitro, such as by transesterification.

As used herein, an “analogous sequence” is one wherein the function ofthe gene is essentially the same as a reference gene such as, forexample, a 'tesA gene from E. coli. Additionally, analogous genesinclude at least about 20%, for example, at least about 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thesequence of a reference gene or polynucleotide such as, for example, thepolynucleotide or polypeptide sequence of a 'tesA gene or a 'TesAthioesterase, respectively. In additional embodiments more than one ofthe above properties applies to the sequence. Analogous sequences aredetermined by known methods of sequence alignment.

The term “alignment” refers to a method of comparing two or morepolynucleotides or polypeptide sequences for the purpose of determiningtheir relationship to each other. Alignments are typically performed bycomputer programs that apply various algorithms, however it is alsopossible to perform an alignment by hand. Alignment programs typicallyiterate through potential alignments of sequences and score thealignments using substitution tables, employing a variety of strategiesto reach a potential optimal alignment score. Commonly-used alignmentalgorithms include, but are not limited to, CLUSTALW, (see, Thompson J.D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice, Nucleic AcidsResearch 22: 4673-4680, 1994); CLUSTALV, (see, Larkin M. A., et al.,CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics 23(21):2947-2948, 2007); Jotun-Hein, Muscle et al., MUSCLE: a multiple sequencealignment method with reduced time and space complexity, BMCBioinformatics 5: 113, 2004); Mafft, Kalign, ProbCons, and T-Coffee (seeNotredame et al., T-Coffee: A novel method for multiple sequencealignments, Journal of Molecular Biology 302: 205-217, 2000). Exemplaryprograms that implement one or more of the above algorithms include, butare not limited to MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St.Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView,Phylip, and Discovery Studio from Accelrys(Accelrys, Inc., 10188 TelesisCt, Suite 100, San Diego, Calif. 92121). In a non-limiting example,MegAlign is used to implement the CLUSTALW alignment algorithm with thefollowing parameters: Gap Penalty 10, Gap Length Penalty 0.20, DelayDivergent Seqs (30%) DNA Transition Weight 0.50, Protein Weight matrixGonnet Series, DNA Weight Matrix IUB.

The term “antibodies” refers to immunoglobulins. Antibodies include butare not limited to immunoglobulins obtained directly from any speciesfrom which it is desirable to produce antibodies. In addition, thepresent invention encompasses modified antibodies. The term also refersto antibody fragments that retain the ability to bind to the sameepitope to which the intact antibody also binds, and include polyclonalantibodies, monoclonal antibodies, chimeric antibodies, anti-idiotype(anti-ID) antibodies. Antibody fragments include, but are not limited tothe complementarity-determining regions (CDRs), single-chain fragmentvariable regions (scFv), heavy chain variable region (VH), light chainvariable region (VL). Polyclonal and monoclonal antibodies are alsoencompassed by the present invention. Preferably, the antibodies aremonoclonal antibodies.

The term “attenuate” means to weaken, reduce or diminish. In oneexample, the sensitivity of a particular enzyme to feedback inhibitionor inhibition caused by a composition that is not a product or areactant (non-pathway specific feedback) is reduced such that the enzymeactivity is not impacted by the presence of a compound. In a particularexample, the expression of fabH gene is temperature sensitive and itssequence can be altered to decrease the sensitivity to temperaturefluctuations. Also, expression of the fabH gene can be attenuated whenbranched amino acids are desired. In another example, an enzyme that hasbeen modified to be less active can be referred to as attenuated. Afunctional modification of the sequence encoding an enzyme can be usedto attenuate expression of an enzyme. Sequence modifications mayinclude, for example, a mutation, deletion, or insertion of one or morenucleotides in a gene sequence or a sequence controlling thetranscription or translation of a gene sequence, which modificationresults in a reduction or inhibition of production of the gene product,or renders the gene product non-functional. For example, functionaldeletion of fabR in E. coli reduces the repression of the fatty acidbiosynthetic pathway and allows E. coli to produce more unsaturatedfatty acids (UFAs). In some instances a functional deletion is describedas a knock-out mutation. Other methods are available for attenuatingexpression of an enzyme. For example, attenuation can be accomplished bymodifying the sequence encoding the gene as described above; placing thegene under the control of a less active promoter, expressing interferingRNAs, ribozymes, or antisense sequences that target the gene ofinterest; by changing the physical or chemical environment, such astemperature, pH, or solute concentration, such that the optimal activityof the gene or gene product is not realized; or through any othertechniques known in the art.

The term “biocrude” refers to a biofuel that can be used as a substituteof petroleum-based fuels. In addition, biocrude, like petroleum crude,can be converted into other fuels, for example gasoline, diesel, jetfuel, or heating oil. Moreover, biocrude, like petroleum crude, can beconverted into other industrially useful chemicals for use in, forexample, pharmaceuticals, cosmetics, consumer goods, industrialprocesses, etc. A biocrude composition can comprise, for example,hydrocarbons, hydrocarbon products, fatty acid esters, and/or aliphaticketones, or a combination thereof. In a preferred embodiment, a biocrudecomposition is comprised of hydrocarbons, for example, aliphatic (e.g.,alkanes, alkenes, alkynes) or aromatic hydrocarbons.

The term “biodiesel” refers to a particular kind of biofuel that can beused in diesel engines. Biodiesel can be a substitute for traditionaldiesel, which is typically derived from petroleum. Biodiesel can be usedin internal combustion diesel engines in either a pure form, which isreferred to as “neat” biodiesel, or as a mixture in any concentrationwith a petroleum-based diesel. A biodiesel composition can also comprisevarious suitable additives. Biodiesel can be comprised of hydrocarbonsor esters. In one embodiment, biodiesel is comprised of fatty esters,such as fatty acid methyl esters (FAME) or fatty acid ethyl esters(FAEE). In a preferred embodiment, these FAME and FAEE are comprised offatty acyl moieties having a carbon chain length of about 8-20, 10-18,or 12-16. Fatty esters used as biodiesel may contain carbon chains thatare straight, branched, saturated, or unsaturated.

The term “biofuel” refers to any fuel derived from biomass. Biomass is abiological material that can be converted into a biofuel. One exemplarysource of biomass is plant matter. For example, corn, sugar cane, andswitchgrass can be used as biomass. Another non-limiting example ofbiomass is animal matter, for example cow manure. Biomass also includeswaste products from industry, agriculture, forestry, and households.Examples of such waste products include, without limitation,fermentation waste, straw, lumber, sewage, garbage and food leftoversand glycerol. Biomass also includes sources of carbon, such ascarbohydrates (e.g., sugars). Biofuels can be substituted for petroleumbased fuels. For example, biofuels are inclusive of transportation fuels(e.g., gasoline, diesel, jet fuel, etc.), heating fuels, andelectricity-generating fuels. A biofuel is a renewable energy source.Non-limiting examples of biofuels include biodiesel, hydrocarbons (e.g.,alkanes, alkenes, alkynes, or aromatic hydrocarbons), and alcoholsderived from biomass.

The term “carbon chain length” is defined herein as the number of carbonatoms in a carbon chain of a thioesterase substrate or a fatty acidderivative. The carbon chain length of a particular molecule is markedas C_(x), wherein the subscript “x” refers to the number of carbons inthe carbon chain. As used herein, the term “long-chain” refers to thosemolecules that have a carbon chain of about 15 to about 20 carbons long(e.g., C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀). The term “short-chain” refersto those molecules that have a carbon chain of about 8 to about 14carbons long (e.g., C₈, C₉, C₁₀, C₁₁, or C₁₂).

The term “carbon source” means a substrate or compound suitable to beused as a source of carbon for prokaryotic or simple eukaryotic cellgrowth. Carbon sources can be in various forms, including, but notlimited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones,amino acids, peptides, gases (e.g., CO and CO₂), and the like. Theseinclude, for example, various monosaccharides such as glucose, fructose,mannose and galactose; oligosaccharides such as fructo-oligosaccharideand galacto-oligosaccharide; polysaccharides such as xylose, andarabinose; disaccharides such as sucrose, maltose and turanose;cellulosic material such as methyl cellulose and sodium carboxymethylcellulose; saturated or unsaturated fatty acid esters such as succinate,lactate and acetate; alcohols such as ethanol, etc., or mixturesthereof. The carbon source can additionally be a product ofphotosynthesis, including, but not limited to glucose. Glycerol can bean effective carbon source as well. Suitable carbon sources can begenerated from any number of natural and renewable sources, includingparticularly biomass from agricultural, municipal and industrial waste,so long as the material can be used as a component of a fermentation toprovide a carbon source. Biomass sources include corn stover, sugarcane,switchgrass, animal matter, or waste materials.

The term “chromosomal integration” means the process whereby an incomingsequence is introduced into the chromosome of a host cell. Thehomologous regions of the transforming DNA align with homologous regionsof the chromosome. Then, the sequence between the homology boxes can bereplaced by the incoming sequence in a double crossover (i.e.,homologous recombination). In some embodiments of the present invention,homologous sections of an inactivating chromosomal segment of a DNAconstruct align with the flanking homologous regions of the indigenouschromosomal region of the microbial chromosome. Subsequently, theindigenous chromosomal region is deleted by the DNA construct in adouble crossover.

The term “cloud point” refers to the temperature of a liquid at whichthe dissolved solids are no longer completely soluble, precipitating asa second phase and giving the fluid a cloudy appearance. This term isrelevant to a number of applications with somewhat or completelydifferent consequences. In the petroleum industry, cloud point refers tothe temperature below which wax or other heavy hydrocarbons crystalizein a crude oil, refined oil or fuel to form a cloudy appearance. Thepresence of solidified wax influences the flowing behavior of the fluid,raising the tendency to clog fuel filters/injectors and other machineparts, causing accumulation of wax on cold surfaces (e.g., on pipelinesurfaces or heat exchanger surfaces), and changing even the emulsioncharacteristics with water. Cloud point is an indication of the tendencyof the oil to plug filters or small orifices at cold operatingtemperatures. The cloud point of a nonionic surfactant or glycolsolution is the temperature at which the mixture starts to separate intotwo or more phases, thus becoming cloudy. This behavior ischaracteristic of non-ionic surfactants containing polyoxyethylenechains, which can exhibit reverse solubility versus temperature behaviorin water, and therefore can “cloud out” at some point as the temperatureis raised. Glycols demonstrating this behavior are known as “cloud-pointglycols” and are used as shale inhibitors. The cloud point is typicallyalso affected by salinity, being generally lower in more saline fluids.

The term “cloud point lowering additive” refers to an additive that canbe added to a composition to decrease or lower the cloud point of thecomposition, as described above.

The term “conditions that permit product production” refers to anyfermentation conditions that allow a production host to produce adesired product, such as acyl-CoA or fatty acid derivatives including,for example, fatty acids, hydrocarbons, fatty alcohols, waxes, or fattyesters. Fermentation conditions usually comprise many parameters.Exemplary conditions include, but are not limited to, temperatureranges, levels of aeration, pH ranges, and media composition (e.g.,solvents and solutes). Each of these conditions, individually and incombination, allows the production host to grow. Exemplary media includebroths or gels. Generally, a suitable medium includes a carbon source,such as glucose, fructose, cellulose, or the like, which can bemetabolized by the microorganism directly. In addition, enzymes can beused in the medium to facilitate the mobilization (e.g., thedepolymerization of starch or cellulose to fermentable sugars) andsubsequent metabolism of the carbon source. To determine if the cultureconditions are suitable for product production, the production host canbe cultured for about 4, 8, 12, 24, 36, 48, or 72 hours. Duringculturing or after culturing, samples can be obtained and analyzed todetermine if the culture conditions permit product production. Forexample, the production hosts in the sample or the medium in which theproduction hosts were grown can be tested for the presence of thedesired product. When testing for the presence of a product, assays,such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, aswell as those provided in the examples herein, can be used.

The term “consensus sequence” or “canonical sequence” refers to anarchetypical amino acid sequence against which all variants of aparticular protein or sequence of interest are compared. Either termalso refers to a sequence that sets forth the nucleotides that are mostoften present in a polynucleotide sequence of interest. For eachposition of a protein, the consensus sequence gives the amino acid thatis most abundant in that position in the sequence alignment.

As used herein, the term “consensus mutation” refers to a difference inthe sequence of a starting gene and a consensus sequence. Consensusmutations are identified by comparing the sequences of the starting geneand the consensus sequence resulting from a sequence alignment. In someembodiments, consensus mutations are introduced into the starting genesuch that it becomes more similar to the consensus sequence. Consensusmutations also include amino acid changes that change an amino acid in astarting gene to an amino acid that is more frequently found in amultiple sequence alignment (MSA) at that position relative to thefrequency of that amino acid in the starting gene. Thus, the term“consensus mutation” refers to any amino acid change that replaces anamino acid of the starting gene with an amino acid that is more abundantin the MSA than the native amino acid.

The term “conservative substitutions” or “conserved substitutions”refers to, for example, a substitution wherein one or more of thefollowing amino acid substitutions are made: replacement of an aliphaticamino acid, such as alanine, valine, leucine, and isoleucine, withanother aliphatic amino acid; replacement of a serine with a threonine;replacement of a threonine with a serine; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as histidine, lysine and arginine,with another basic residue; and replacement of an aromatic residue, suchas tryptophan, phenylalanine and tyrosine, with another aromaticresidue; or replacement of small amino acids, such as glycine, alanine,serine, threonine and methionine, with another small amino acid. Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, in The Proteins, Academic Press, New York, 1979. Usefulconservative modifications include Alanine to Cysteine, Glycine, orSerine; Arginine to Isoleucine, Lysine, Methionine, or Ornithin;Asparagine to Aspartic acid, Glutamine, Glutamic acid, or Histidine;Aspartic acid to Asparagine, Glutamine, or Glutamic acid; Cysteine toMethionine, Serine, or Threonine; Glutamine to Asparagine, Asparticacid, or Glutamic acid; Glutamic acid to Asparagine, Aspartic acid, orGlatmine; Glycine to Aspartic acid, Alanine, or Proline; Histidine toAsparagine, or Glutamine; Isoleucine to Leucine, Methionine, or Valine;Leucine to Isoleucine, Methionine, or Valine; Lysine to Arginine,Glutamine, Glutamic acid, Isoleucine, Methionine, or Ornithin;Methionine to Cysteine, Isoleucine, Leucine, or Valine; Phenylalanine toHistidine, L-Dopa, Leucine, Methionine, Threonine, Tryptophan, Tyrosine,3-phenylproline, 4-phenylproline, or 5-phenylproline; Proline toL-1-thioazolidine-4-carboxylic acid or D- orL-1-oxazolidine-4-carboxylic acid; Serine to Cysteine, Methionine, orThreonine; Threonine to Methionine, Serine, or Valine; Tryptophan toTyrosine; Tyrosine to L-Dopa, Histidine, or Phenylalanine; and Valine toIsoleucine, Leucine, or Methionine.

The term “corresponds to” refers to an amino acid residue in a firstprotein sequence being positionally equivalent to an amino acid residuein a second reference protein sequence by virtue of the fact that theresidue in the first protein sequence lines up with the residue in thereference sequence using bioinformatic techniques, for example, usingthe methods described herein for preparing a sequence alignment. Thecorresponding residue in the first protein sequence is then assigned theresidue number in the second reference protein sequence. The firstprotein sequence can be analogous to the second protein sequence ornon-analogous to the second protein sequence, although it is preferredthat the two protein sequences are analogous sequences. For example,when the amino acid sequence of an E. coli 'TesA, SEQ ID NO:31 in FIG.58, is used as a reference sequence, each of the amino acid residues inanother aligned protein of interest or an analogous protein can beassigned a residue number corresponding to the residue numbers 1-182 ofSEQ ID NO:31. For example, in FIG. 47, the aligned amino acid sequencesare referenced or corresponded to the sequence of an E. coli 'TesA.Accordingly, a given position in another thioesterase of interest,either a precursor or a mutant thioesterase, can be assigned acorresponding position in the 'TesA sequence, using known bioinformatictechniques such as those described herein.

The term “deletion,” when used in the context of an amino acid sequence,means a deletion in or a removal of a residue from the amino acidsequence of a precursor protein, resulting in a mutant protein havingone less amino acid residue as compared to the precursor protein. Theterm can also be used in the context of a nucleotide sequence, whichmeans a deletion in or removal of a residue from the polynucleotidesequence of a precursor polynucleotide.

The term “derived from” and “obtained from” refer to, in the context ofa precursor thioesterase, a thioesterase produced or producible by astrain of the organism in question, and also a thioesterase encoded by apolynucleotide sequence isolated from such strain and produced in a hostorganism containing such a polynucleotide sequence. Additionally, theterms refer to a thioesterase that is encoded by a polynucleotidesequence of synthetic and/or cDNA origin and that has the identifyingcharacteristics of the thioesterase in question. To exemplify,“thioesterases derived from Enterobacteriacaea” refers to those enzymeshaving thioesterase activity that are naturally produced byEnterobacteriacaea, as well as to thioesterases like those produced byEnterobacteriacaea sources but that, through the use of geneticengineering techniques, are produced by non-Enterobacteriocaea organismstransformed with a polynucleotide encoding said thioesterase.

The term “DNA construct” and “transforming DNA” are used interchangeablyherein to refer to a DNA used to introduce sequences into a host cell ororganism. Typically a DNA construct is generated in vitro by PCR orother suitable technique(s) known to those in the art. In certainembodiments, the DNA construct comprises a sequence of interest (e.g.,an incoming sequence). In some embodiments, the sequence is operablylinked to additional elements such as control elements (e.g., promoters,etc.). A DNA construct can further comprise a selectable marker. It canalso comprise an incoming sequence flanked by homology boxes. In afurther embodiment, the DNA construct comprises other non-homologoussequences, added to the ends (e.g., stuffer sequences or flanks). Insome embodiments, the ends of the incoming sequence are closed such thatthe DNA construct forms a closed circle. The transforming sequences maybe wildtype, mutant or modified. In some embodiments, the DNA constructcomprises sequences homologous to the host cell chromosome. In otherembodiments, the DNA construct comprises non-homologous sequences. Oncethe DNA construct is assembled in vitro it may be used to: 1) insertheterologous sequences into a desired target sequence of a host cell; 2)mutagenize a region of the host cell chromosome (i.e., replace anendogenous sequence with a heterologous sequence); 3) delete targetgenes; and/or (4) introduce a replicating plasmid into the host.

A polynucleotide is said to “encode” an RNA or a polypeptide if, in itsnative state or when manipulated by methods known to those of skill inthe art, it can be transcribed and/or translated to produce the RNA, thepolypeptide, or a fragment thereof. The antisense strand of such apolynucleotide is also said to encode the RNA or polypeptide sequences.As is known in the art, a DNA can be transcribed by an RNA polymerase toproduce an RNA, and an RNA can be reverse transcribed by reversetranscriptase to produce a DNA. Thus a DNA can encode an RNA, and viceversa.

The phrase “equivalent,” in this context, refers to thioesterase enzymesthat are encoded by a polynucleotide capable of hybridizing to thepolynucleotide having the sequence of SEQ ID NO:31 in FIG. 58, underconditions of medium to maximum stringency. For example, beingequivalent means that an equivalent mature thioesterase comprises atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, and/or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO:31 in FIG. 58.

An “ester synthase” is a peptide capable of catalyzing a biochemicalreaction to producing esters. For example, an ester synthase is apeptide that is capable of participating in converting a thioester to afatty ester. In certain embodiments, an ester synthase converts athioester, acyl-CoA, to a fatty ester. In an alternate embodiment, anester synthase uses a thioester and an alcohol as substrates to producea fatty ester. Ester synthases are capable of using short and long chainacyl-COAs as substrates. In addition, ester synthases are capable ofusing short and long chain alcohols as substrates. Non-limiting examplesof ester synthases include wax synthases, wax-ester synthases,acyl-CoA:alcohol transacylases, acyltransferases, fatty acyl-coenzymeA:fatty alcohol acyltransferases, fatty acyl-ACP transacylase, andalcohol acetyltransferase. An ester synthase that converts an acyl-CoAthioester to a wax is called a wax synthase. Exemplary ester synthasesinclude those classified under the enzyme classification number EC2.3.1.75. The term “ester synthase” does not comprise enzymes that alsohave thioesterase activity. The ones that have both ester synthaseactivity and thioesterase activity are categorized as thioesterasesherein.

The term “expressed genes” refers to genes that are transcribed intomessenger RNA (mRNA) and then translated into protein, as well as genesthat are transcribed into types of RNA, such as transfer RNA (tRNA),ribosomal RNA (rRNA), and regulatory RNA, which are not translated intoprotein.

The terms “expression cassette” or “expression vector” refers to apolynucleotide construct generated recombinantly or synthetically, witha series of specified elements that permit transcription of a particularpolynucleotide in a target cell. A recombinant expression cassette canbe incorporated into a plasmid, chromosome, mitochondrial DNA, plasmidDNA, virus, or polynucleotide fragment. Typically, the recombinantexpression cassette portion of an expression vector includes, amongother sequences, a polynucleotide sequence to be transcribed and apromoter. In particular embodiments, expression vectors have the abilityto incorporate and express heterologous polynucleotide fragments in ahost cell. Many prokaryotic and eukaryotic expression vectors arecommercially available. Selection of appropriate expression vectors iswithin the knowledge of those of skill in the art. The term “expressioncassette” is also used interchangeably herein with “DNA construct,” andtheir grammatical equivalents.

The term “fatty acid derivative,” as used herein, refers to acomposition that is derived from a metabolic pathway, which pathwayincludes a thioesterase reaction. Thus, fatty acid derivative productscan be products that are, or are derived from, fatty acid or fattyesters that are products of a thioesterase reaction. Fatty acidderivatives thus include, for example, products that are, or that arederived from, fatty acids that are the direct reaction product of athioesterase, and/or a fatty ester that is a direct reaction product ofa thioesterase. Exemplary fatty acid derivatives include, for example,short and long chain alcohols, hydrocarbons, and fatty alcohols andesters, including waxes, fatty acid esters, and/or fatty esters.Specific non-limiting examples of fatty acid derivatives include fattyacids, fatty acid methyl esters, fatty acid ethyl esters, fattyalcohols, fatty alkyl-acetates, fatty aldehydes, fatty amines, fattyamides, fatty sulfates, fatty ethers, ketones, alkanes, internalolefins, terminal olefins, dicarboxylic acids, ω-dicarboxylic acids,diols and terminal and/or internal fatty acids.

The term “fatty acid derivative enzymes” refers to, collectively andindividually, enzymes that may be expressed or overexpressed in theproduction of fatty acid derivatives. These enzymes may be parts of afatty acid biosynthetic pathway. Non-limiting examples of fatty acidderivative synthases include fatty acid synthases, thioesterases,acyl-CoA synthases, acyl-CoA reductases, alcohol dehydrogenases, alcoholacyltransferases, fatty alcohol-forming acyl-CoA reductase, fatty aciddecarbonylases, carboxylic acid reductases, fatty alcohol acetyltransferases, and ester synthases. Fatty acid derivative enzymes convertsubstrates into fatty acid derivatives. In certain circumstances, asuitable substrate may be a first fatty acid derivative, which isconverted by a fatty acid derivative enzyme into a different, secondfatty acid derivative.

The term “fatty alcohol” refers to an alcohol having the formula ROH. Incertain embodiments, a fatty alcohol is an alcohol made from a fattyacid or fatty acid derivative. In one embodiment, the R group is atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20carbons in length. R can be straight or branched chain. The branchedchains may have one or more points of branching. In addition, thebranched chains may include cyclic branches, such as cyclopropane orepoxide moieties. Furthermore, R can be saturated or unsaturated. Ifunsaturated, R can have one or more points of unsaturation. In oneembodiment, the fatty alcohol is produced biosynthetically. Fattyalcohols have many uses. For example, fatty alcohols can be used toproduce specialty chemicals. Specifically, fatty alcohols can be used asbiofuels; as solvents for fats, waxes, gums, and resins; inpharmaceutical salves, emollients and lotions; as lubricating-oiladditives; in detergents and emulsifiers; as textile antistatic andfinishing agents; as plasticizers; as nonionic surfactants; and incosmetics, for example as thickeners.

The term“fatty alcohol forming peptides” refers to peptides capable ofcatalyzing the conversion of acyl-CoA to fatty alcohol, including fattyalcohol forming acyl-CoA reductase (FAR, EC 1.1.1.*), acyl-CoA reductase(EC 1.2.1.50) or alcohol dehydrogenase (EC 1.1.1.1). Additionally, oneof ordinary skill in the art will appreciate that some fatty alcoholforming peptides will catalyze other reactions as well. For example,some acyl-CoA reductase peptides will accept substrates other than fattyacids. Such non-specific peptides are, therefore, also included.Polynucleotide sequences encoding fatty alcohol forming peptides areknown in the art and such peptides are publicly available.

The term “fatty aldehyde” refers to an aldehyde having the formula RCHOcharacterized by an unsaturated carbonyl group (C═O). In certainembodiments, a fatty aldehyde is an aldehyde made from a fatty acid orfatty acid derivative. In one embodiment, the R group is at least about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20carbons in length. R can be straight or branched chain. The branchedchains may have one or more points of branching. In addition, thebranched chains can be cyclic branches. Furthermore, R can be saturatedor unsaturated. If unsaturated, R can have one or more points ofunsaturation. In one embodiment, the fatty aldehyde is producedbiosynthetically. Fatty aldehydes have many uses. For example, fattyaldehydes can be used to produce specialty chemicals. Specifically,fatty aldehydes can be used to produce polymers, resins, dyes,flavorings, plasticizers, perfumes, pharmaceuticals, and otherchemicals. Some are used as solvents, preservatives, or disinfectants.Some natural and synthetic compounds, such as vitamins and hormones, arealso aldehydes.

The terms “fatty aldehyde biosynthetic polypeptide,” “carboxylic acidreductase,” and “CAR” are used interchangeably herein.

The term “fatty ester” refers to an ester having greater than 5 carbonatoms. In certain embodiments, a fatty ester is an ester made from afatty acid, for example a fatty acid ester. In one embodiment, a fattyester contains an A side (i.e., the carbon chain attached to thecarboxylate oxygen) and a B side (i.e., the carbon chain comprising theparent carboxylate). In a particular embodiment, when a fatty ester isderived from the fatty acid biosynthetic pathway, the A side iscontributed by an alcohol, and the B side is contributed by a fattyacid. Any alcohol can be used to form the A side of the fatty esters.For example, the alcohol can be derived from the fatty acid biosyntheticpathway. Alternatively, the alcohol can be produced through non-fattyacid biosynthetic pathways. Moreover, the alcohol can be providedexogenously. For example, the alcohol can be supplied to thefermentation broth in instances where the fatty ester is produced by anorganism. Alternatively, a carboxylic acid, such as a fatty acid oracetic acid, can be supplied exogenously in instances where the fattyester is produced by an organism that can also produce alcohol. Thecarbon chains comprising the A side or B side can be of any length. Inone embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5,6, 7, 8, 10, 12, 14, 16, 18, or 20 carbons in length. The B side of theester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26carbons in length. The A side and/or the B side can be straight orbranched chain. The branched chains may have one or more points ofbranching. In addition, the branched chains may include cyclic branches,such as cyclopropane or epoxide moieties. Furthermore, the A side and/orB side can be saturated or unsaturated. If unsaturated, the A sideand/or B side can have one or more points of unsaturation. In oneembodiment, the fatty ester is produced biosynthetically. In thisembodiment, first the fatty acid is “activated.” Non-limiting examplesof activated fatty acids are acyl-CoA, acyl ACP, acyl-AMP, and acylphosphate. Acyl-CoA can be a direct product of fatty acid biosynthesisor degradation. In addition, acyl-CoA can be synthesized from a freefatty acid, a CoA, and an adenosine nucleotide triphosphate (ATP). Anexample of an enzyme that produces acyl-CoA is an acyl-CoA synthase.After the fatty acid is activated, it can be readily transferred to arecipient nucleophile. Exemplary nucleophiles are alcohols, thiols,amines, or phosphates. In another embodiment, the fatty ester can bederived from a fatty acyl-thioester and an alcohol. In one embodiment,the fatty ester is a wax. The wax can be derived from a long chain fattyalcohol and a long chain fatty acid. In another embodiment, the fattyester is a fatty acid thioester, for example fatty acyl Coenzyme A(acyl-CoA). In other embodiments, the fatty ester is a fatty acylpanthothenate, an acyl acyl carrier protein (acyl-ACP), a fatty acylenzyme ester, or a fatty phosphate ester. An ester can be formed from anacyl enzyme ester intermediate through the alcoholysis of the ester bondto form a new ester and the free enzyme. Fatty esters have many uses.For example, fatty esters can be used as, or as a component of, abiofuel or a surfactant.

The term “fatty ester vs. other fatty acid derivatives” as used hereinrefers to the proportional yield of fatty ester in comparison with thetotal amount of other fatty acid derivatives that are not fatty esters.In other words, the amount of fatty esters is compared with the amountof fatty acid derivatives other than fatty esters.

The term “fermentation productivity” or “productivity” refers to therate of product production and is expressed g L⁻¹h⁻¹. SpecificProductivity is the productivity normalized for catalyst concentrationand is expressed as g/g L⁻¹h⁻¹ g (catalyst)⁻¹.

The term “fermentation titer” or “titer” refers to the concentration ofa reaction product, usually expressed as g/L but also in other units(i.e., molar, mass/mass, mass/volume, or volume/volume).

The term “fermentation yield” or “yield” refers to the amount of productproduced from a given amount of raw material and is usually expressed asthe ratio of mass of the product produced divided by the mass of rawmaterial consumed (g product/g raw material). It can also be expressed amolar yield (moles product/moles raw material).

The term “fraction of modern carbon” refers to the parameter “f_(M)” asdefined by National Institute of Standards and Technology (NIST)Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalicacids standards HOxI and HOxII, respectively. The fundamental definitionrelates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD1950). This is roughly equivalent to decay-corrected pre-IndustrialRevolution wood. For the current living biosphere (plant material),f_(M) is about 1.1.

The term “functional assay” refers to an assay that provides anindication of a protein's activity. In particularly preferredembodiments, the term refers to an assay system in which a protein isanalyzed for its ability to function in its natural capacity. Forexample, in the case of enzymes, a functional assay involves determiningthe effectiveness of the enzyme in catalyzing a reaction.

“Gene” refers to a polynucleotide (e.g., a DNA segment), which encodes apolypeptide, and includes regions preceding and following the codingregions as well as intervening sequences (introns) between individualcoding segments (exons).

The term “homologous genes” refers to a pair of genes from different butrelated species, which correspond to each other and which are identicalor similar to each other. The term encompasses genes that are separatedby the speciation process during the development of new species) (e.g.,orthologous genes), as well as genes that have been separated by geneticduplication (e.g., paralogous genes).

The term “endogenous protein” refers to a protein that is native to ornaturally occurring in a cell. “Endogeneous polynucleotide” refers to apolynucleotide that is in the cell and was not introduced into the cellusing recombinant engineering techniques. For example, a gene that waspresent in the cell when the cell was originally isolated from nature. Agene is still considered endogenous if the control sequences, such as apromoter or enhancer sequences that activate transcription ortranslation, have been altered through recombinant techniques.Conversely, the term “heterologous” is also used herein, and refers to aprotein or a polynucleotide that does not naturally occur in a hostcell.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules or paired chromosomes at sites ofidentical or nearly identical nucleotide sequences. In certainembodiments, chromosomal integration is homologous recombination.

The term “homologous sequences” as used herein refers to apolynucleotide or polypeptide sequence having, for example, about 100%,about 99% or more, about 98% or more, about 97% or more, about 96% ormore, about 95% or more, about 94% or more, about 93% or more, about 92%or more, about 91% or more, about 90% or more, about 88% or more, about85% or more, about 80% or more, about 75% or more, about 70% or more,about 65% or more, about 60% or more, about 55% or more, about 50% ormore, about 45% or more, or about 40% or more sequence identity toanother polynucleotide or polypeptide sequence when optimally alignedfor comparison. In particular embodiments, homologous sequences canretain the same type and/or level of a particular activity of interest.In some embodiments, homologous sequences have between 85% and 100%sequence identity, whereas in other embodiments there is between 90% and100% sequence identity. In particular embodiments, there is 95% and 100%sequence identity.

“Homology” refers to sequence similarity or sequence identity. Homologyis determined using standard techniques known in the art (see, e.g.,Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch,J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.USA 85:2444, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package (Genetics Computer Group,Madison, Wis.); and Devereux et al., Nucl. Acid Res., 12:387-395, 1984).A non-limiting example includes the use of the BLAST program (Altschulet al., Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs, Nucleic Acids Res. 25:3389-3402, 1997) to identifysequences that can be said to be “homologous.” A recent version such asversion 2.2.16, 2.2.17, 2.2.18, 2.2.19, or the latest version, includingsub-programs such as blastp for protein-protein comparisons, blastn fornucleotide-nucleotide comparisons, tblastn for protein-nucleotidecomparisons, or blastx for nucleotide-protein comparisons, and withparameters as follows: Maximum number of sequences returned 10,000 or100,000; E-value (expectation value) of 1e-2 or 1e-5, word size 3,scoring matrix BLOSUM62, gap cost existence 11, gap cost extension 1,may be suitable. An E-value of 1e-5, for example, indicates that thechance of a homologous match occurring at random is about 1 in 10,000,thereby marking a high confidence of true homology.

The term “host strain” or “host cell” refers to a suitable host for anexpression vector comprising a DNA of the present invention.

The term “hybridization” refers to the process by which a strand ofpolynucleotide joins with a complementary strand through base pairing,as known in the art. A polynucleotide sequence is considered to be“selectively hybridizable” to a reference polynucleotide sequence if thetwo sequences specifically hybridize to one another under moderate tohigh stringency hybridization and wash conditions. Hybridizationconditions are based on the melting temperature (T_(m)) of thepolynucleotide binding complex or probe. For example, “maximumstringency” typically occurs at about T_(m)−5° C. (5° C. below the T_(m)of the probe); “high stringency” at about 5-10° C. below the T_(m);“intermediate stringency” at about 10-20° C. below the T_(m) of theprobe; and “low stringency” at about 20-25° C. below the T_(m).Functionally, maximum stringency conditions may be used to identifysequences having strict identity or near-strict identity with thehybridization probe; while an intermediate or a low stringencyhybridization can be used to identify or detect polynucleotide sequencehomologs. Moderate and high stringency hybridization conditions are wellknown in the art. An example of high stringency conditions includeshybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/mL denatured carrier DNA followed bywashing two times in 2×SSC and 0.5% SDS at room temperature and twoadditional times in 0.1×SSC and 0.5% SDS at 42° C. An example ofmoderate stringent conditions includes an overnight incubation at 37° C.in a solution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate and 20 mg/mL denaturated sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37° C. toabout 50° C. Those of skill in the art know how to adjust thetemperature, ionic strength, and other conditions as necessary toaccommodate factors such as probe length and the like.

The term “hydrocarbon” refers to chemical compounds that contain theelements carbon (C) and hydrogen (H). All hydrocarbons consist of acarbon backbone and atoms of hydrogen attached to that backbone.Sometimes, the term is used as a shortened form of the term “aliphatichydrocarbon.” There are essentially three types of hydrocarbons: (1)aromatic hydrocarbons, which have at least about one aromatic ring; (2)saturated hydrocarbons, also known as alkanes, which lack double, tripleor aromatic bonds; and (3) unsaturated hydrocarbons, which have one ormore double or triple bonds between carbon atoms and include, forexample, alkenes (e.g., dienes), and alkynes.

The term “identical,” in the context of two polynucleotide orpolypeptide sequences, means that the residues in the two sequences arethe same when aligned for maximum correspondence, as measured using asequence comparison or analysis algorithm such as those describedherein. For example, if when properly aligned, the correspondingsegments of two sequences have identical residues at 5 positions out of10, it is said that the two sequences have a 50% identity. Mostbioinformatic programs report percent identity over aligned sequenceregions, which are typically not the entire molecules. If an alignmentis long enough and contains enough identical residues, an expectationvalue can be calculated, which indicates that the level of identity inthe alignment is unlikely to occur by random chance.

The term “improving mutation” or “performance-enhancing mutation” refersto a mutation in a protein that lead to altered properties, which conferimproved performance in terms of a target and/or desired property of aprotein as compared to a precursor protein.

The term “insertion,” when used in the context of a polypeptidesequence, refers to an insertion in the amino acid sequence of aprecursor polypeptide, resulting in a mutant polypeptide having an aminoacid that is inserted between two existing contiguous amino acids, i.e.,adjacent amino acids residues, which are present in the precursorpolypeptide. The term “insertion,” when used in the context of apolynucleotide sequence, refers to an insertion of one or morenucleotides in the precursor polynucleotide between two existingcontiguous nucleotides, i.e., adjacent nucleotides, which are present inthe precursor polynucleotides.

The term “introduced” refers to, in the context of introducing apolynucleotide sequence into a cell, any method suitable fortransferring the polynucleotide sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, conjugation, and transduction (see, e.g.,Ferrari et al., Genetics, in Hardwood et al, (eds.), Bacillus, PlenumPublishing Corp., pp. 57-72, 1989).

The term “isolated” or “purified” means a material that is removed fromits original environment, for example, the natural environment if it isnaturally occurring, or a fermentation broth if it is produced in arecombinant host cell fermentation medium. A material is said to be“purified” when it is present in a particular composition in a higher orlower concentration than the concentration that exists prior to thepurification step(s). For example, with respect to a compositionnormally found in a naturally-occurring or wild type organism, such acomposition is “purified” when the final composition does not includesome material from the original matrix. As another example, where acomposition is found in combination with other components in arecombinant host cell fermentation medium, that composition is purifiedwhen the fermentation medium is treated in a way to remove somecomponent of the fermentation, for example, cell debris or otherfermentation products, through, for example, centrifugation ordistillation. As another example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, whether suchprocess is through genetic engineering or mechanical separation. Suchpolynucleotides can be parts of vectors. Alternatively, suchpolynucleotides or polypeptides can be parts of compositions. Suchpolynucleotides or polypeptides can be considered “isolated” because thevectors or compositions comprising thereof are not part of their naturalenvironments. In another example, a polynucleotide or protein is said tobe purified if it gives rise to essentially one band in anelectrophoretic gel or a blot.

The term “mature,” in the context of a protein, means a form of aprotein or peptide that is in its final functional form. To exemplify, amature form of a thioesterase of the present invention comprises theamino acid residues 1-182 of SEQ ID NO:31 in FIG. 58.

The term “modified fatty acid derivatives” refers to products made, atleast in part, from a part of the fatty acid biosynthetic pathway of arecombinant host cell, wherein the product differs from the product madeby such host cell in the absence of the mutant thioesterase of theinvention. Thus, where a mutant thioesterase (or naturally-occurringequivalent thereof) is introduced into a recombinant host cell,resulting in the production of a fatty acid derivative that has adifferent product profile, for example, a higher or lower concentrationof certain fatty acid derivatives having a specific chain length, or ahigher or lower concentration of a certain type of fatty acidderivative, that fatty acid material is “modified” within the context ofthis invention.

The term “mutant thioesterase” or “variant thioesterase” refers to athioesterase that comprises a mutation with reference to a precursorthioesterase.

The term “mutation” refers to, in the context of a polynucleotide, amodification to the polynucleotide sequence resulting in a change in thesequence of a polynucleotide with reference to a precursorpolynucleotide sequence. A mutant polynucleotide sequence can refer toan alteration that does not change the encoded amino acid sequence, forexample, with regard to codon optimization for expression purposes, orthat modifies a codon in such a way as to result in a modification ofthe encoded amino acid sequence. Mutations can be introduced into apolynucleotide through any number of methods known to those of ordinaryskill in the art, including random mutagenesis, site-specificmutagenesis, oligonucleotide directed mutagenesis, gene shuffling,directed evolution techniques, combinatorial mutagenesis, sitesaturation mutagenesis among others.

“Mutation” or “mutated” means, in the context of a protein, amodification to the amino acid sequence resulting in a change in thesequence of a protein with reference to a precursor protein sequence. Amutation can refer to a substitution of one amino acid with anotheramino acid, an insertion or a deletion of one or more amino acidresidues. Specifically, a mutation can also be the replacement of anamino acid with a non-natural amino acid, or with a chemically-modifiedamino acid or like residues. A mutation can also be a truncation (e.g.,a deletion or interruption) in a sequence or a subsequence from theprecursor sequence. A mutation may also be an addition of a subsequence(e.g., two or more amino acids in a stretch, which are inserted betweentwo contiguous amino acids in a precursor protein sequence) within aprotein, or at either terminal end of a protein, thereby increasing thelength of (or elongating) the protein. A mutation can be made bymodifying the DNA sequence corresponding to the precursor protein.Mutations can be introduced into a protein sequence by known methods inthe art, for example, by creating synthetic DNA sequences that encodethe mutation with reference to precursor proteins, or chemicallyaltering the protein itself. A “mutant” as used herein is a proteincomprising a mutation. For example, it is also possible to make a mutantby replacing a portion of a thioesterase with a wild type sequence thatcorresponds to such portion but includes a desired variation at aspecific position that is naturally-occurring in the wild type sequence.

A “naturally-occurring equivalent,” in the context of the presentinvention, refers to a naturally-occurring thioesterase, or a portionthereof, that comprises a naturally-occurring residue, wherein thenaturally-occurring residue corresponds to a mutation in 'TesA (e.g., amutation in SEQ ID NO:31 of FIG. 58) that has introduced a desirablealtered property to 'TesA. Examples of naturally-occurring equivalentthioesterases having such modifications are provided in FIG. 55.

The term “operably linked,” in the context of a polynucleotide sequence,refers to the placement of one polynucleotide sequence into a functionalrelationship with another polynucleotide sequence. For example, a DNAencoding a secretory leader (e.g., a signal peptide) is operably linkedto a DNA encoding a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide. A promoter or anenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence. A ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in the same reading frame.

The term “operon region” refers to a group of contiguous genes that aretranscribed as a single transcription unit from a common promoter, andare thereby subject to co-regulation. In some embodiments, the operonincludes a regulator gene.

The term “optimal alignment” refers to the alignment giving the highestoverall alignment score.

The term “orthologs” or “orthologous genes” refers to genes in differentspecies that have evolved from a common ancestral gene by speciation.Typically, orthologs retain the same function during the course ofevolution. Identification of orthologs finds use in the reliableprediction of gene function in newly sequenced genomes.

“Overexpressed” or “overexpression” in a host cell occurs if the enzymeis expressed in the cell at a higher level than the level at which it isexpressed in a corresponding wild-type cell.

The term “paralog” or “paralogous genes” refers to genes that arerelated by duplication within a genome. While orthologs retain the samefunction through the course of evolution, paralogs evolve new functions,even though some functions are often related to the original one.Examples of paralogous genes include, but are not limited to, genesencoding myoglobin and hemoglobin, which arose from the same ancientancestor but evolved to have different functions.

The term “partition coefficient” means the equilibrium concentration ofa compound in an organic phase divided by the concentration atequilibrium in an aqueous phase (e.g., in a fermentation broth). In oneembodiment of the bi-phasic system described herein, the organic phaseis formed by the fatty acid derivative during the production process. Incertain circumstances, an organic phase can also be provided, forexample, a layer of octane can be provided to the fermentation broth tofacilitate product separation. When describing a two phase system, thepartition coefficient, P, is usually discussed in terms of logP. Acompound with a logP of 1 would partition 10:1 to the organic phase. Acompound with a logP of −1 would partition 1:10 to the organic phase. Bychoosing an appropriate fermentation broth and organic phase, a fattyacid derivative with a high logP value will separate into the organicphase even at very low concentrations in the fermentation vessel.

The terms “percent sequence identity,” “percent amino acid sequenceidentity,” “percent gene sequence identity,” and/or “percentpolynucleotide sequence identity,” with respect to two polypeptides,polynucleotides and/or gene sequences (as appropriate), refer to thepercentage of residues that are identical in the two sequences when thesequences are optimally aligned. Thus, 80% amino acid sequence identitymeans that 80% of the amino acids in two optimally aligned polypeptidesequences are identical.

The term “plasmid” refers to a circular double-stranded (ds) DNAconstruct used as a cloning vector, and which forms an extrachromosomalself-replicating genetic element in some eukaryotes or prokaryotes, orintegrates into the host chromosome.

The term “precursor thioesterase” refers a thioesterase protein fromwhich the mutant thioesterase of the invention can be derived, through,for example, recombinant or chemical means. Examples of precursorthioesterases are naturally-occurring or wildtype thioesterases fromplant, animal or microbial sources. A precursor thioesterase can also bea thioesterase that is non-naturally-occurring. An example of anon-naturally-occurring thioesterase is a thioesterase made through, forexample, random mutation, chemical synthesis, molecular evolution, orsite directed mutagenesis, which can serve as a useful starting pointfrom which to design and/or make the mutant thioesterases of theinvention.

A “primer” is an oligonucleotide, whether occurring naturally as in apurified restriction digest sample, or produced synthetically, which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which the synthesis of a primer extension productthat is complementary to a reference polynucleotide strand is induced.Suitable conditions include, for example, the presence of nucleotidesand an inducing agent such as a DNA polymerase, and a suitabletemperature and pH. A primer is preferably single stranded for maximumefficiency in amplification, but can alternatively be double stranded.If double stranded, a primer can be first treated to separate itsstrands before it is used to prepare extension products. In particularembodiments, a primer is an oligodeoxyribonucleotide. In certainpreferred embodiments, a primer is sufficiently long to prime thesynthesis of extension products in the presence of an inducing agent.The exact lengths of primers will depend on a number of factors,including temperature, source of primer, and the methods used foramplification.

The term “probe” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,recombinantly or by PCR amplification, which is capable of hybridizingto another oligonucleotide of interest. A probe may be single-strandedor double-stranded. Probes are useful in the detection, identificationand isolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA or other enzyme-based histochemicalassays), fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

A “production host” is a cell used to produce products. As disclosedherein, a production host is modified to express or overexpress selectedgenes, or to have attenuated expression of selected genes. Non-limitingexamples of production hosts include plant, animal, human, bacteria,yeast, cyanobacteria, algae, and/or filamentous fungi cells.

A “promoter” is a polynucleotide sequence that functions to directtranscription of a downstream gene. In preferred embodiments, thepromoter is appropriate to the host cell in which the target gene isbeing expressed. The promoter, together with other transcriptional andtranslational regulatory polynucleotide sequences (also termed “controlsequences”) is necessary to express a given gene. In general, thetranscriptional and translational regulatory sequences include, but arenot limited to, promoter sequences, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, and enhancer or activator sequences.

The term “promoters” or “enhancers” refers to transcriptional controlsignals in eukaryotes. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription (Maniatis et al., Science, 236:1237, 1987).Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in yeast, insect, mammalian and plantcells. Promoter and enhancer elements have also been isolated fromviruses. Analogous control elements, such as promoters and enhancers,are also found in prokaryotes. The selection of a particular promoterand enhancer depends on the cell type used to express the protein ofinterest. Some eukaryotic and prokaryotic promoters and enhancers have abroad production host cell range while others are functional in alimited subset of production host cells (see, e.g., Voss et al., TrendsBiochem. Sci., 11:287, 1986; Maniatis et al., 1987, supra). The term“promoter element,” “promoter,” or “promoter sequence” refers to a DNAsequence that functions as a switch which activates the expression of agene. If the gene is activated, it is said to be transcribed, orparticipating in transcription. Transcription involves the synthesis ofmRNA from the gene. The promoter, therefore, serves as a transcriptionalregulatory element and also provides a site for initiation oftranscription of the gene into mRNA.

The term “property” refers to, in the context of a polynucleotide, anycharacteristic or attribute of a polynucleotide that can be selected ordetected. These properties include, but are not limited to, a propertyaffecting binding to a polypeptide, a property conferred on a cellcomprising a particular polynucleotide, a property affecting genetranscription (e.g., promoter strength, promoter recognition, promoterregulation, enhancer function), a property affecting RNA processing(e.g., RNA splicing, RNA stability, RNA conformation, andpost-transcriptional modification), a property affecting translation(e.g., level, regulation, binding of mRNA to ribosomal proteins,post-translational modification). For example, a binding site for atranscription factor, polymerase, regulatory factor, and the like, of apolynucleotide may be altered to produce desired characteristics or toidentify undesirable characteristics.

The term “property” refers to, in the context of a protein, anycharacteristic or attribute of a protein that can be selected ordetected.

The terms “protein” and “polypeptide” are used interchangeably herein.The 3-letter code as well as the 1-letter code for amino acid residuesas defined in conformity with the IUPAC-IUB Joint Commission onBiochemical Nomenclature (JCBN) is used throughout this disclosure. Itis also understood that a polypeptide may be coded for by more than onepolynucleotide sequence due to the degeneracy of the genetic code. Anenzyme is a protein.

The terms “proportional yield” and “percentage yield” are usedinterchangeably herein. It refers to the amount of a desired product inrelation to other products that are within the same mixture produced bya recombinant host of the present invention. For example, theproportional yield of a desired product can be improved such that it ismore predominant over the other components in the product mixture toreduce the burden of purification. In another example, the proportionalyield of an undesired product (i.e., a component that will need to beremoved from the desired product) can be reduced such that it is lesspredominant over the desired component in the product mixture to achievethe same end. Proportional yields are expressed herein in the form of “Xvs. other fatty acid derivatives,” which compares the amount of X, whichis a type of fatty acid derivative (e.g., a fatty ester, a fatty acidderivative of a particular chain length), and the term “other fatty acidderivatives” means the aggregate amount of all other fatty acidderivatives other than X that are produced in the same experiment,culture, or fermentation run.

The term “prosequence” refers to an amino acid sequence between thesignal sequence and mature protein that is necessary for the secretionof the protein. Cleavage of the prosequence can lead to a mature activeprotein/enzyme under certain circumstances and suitable conditions.

The term “recombinant,” when used to modify the term “cell” or “vector”herein, refers to a cell or a vector that has been modified by theintroduction of a heterologous polynucleotide sequence, or that the cellis derived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found in identical form within the native(non-recombinant) form of the cells or express, as a result ofdeliberate human intervention, native genes that are otherwiseabnormally expressed, underexpressed or not expressed at all. The terms“recombination,” “recombining,” and generating a “recombined”polynucleotide refer generally to the assembly of two or morepolynucleotide fragments wherein the assembly gives rise to a chimericpolynucleotide made from the assembled parts.

The term “regulatory segment,” “regulatory sequence,” or “expressioncontrol sequence” refers to a polynucleotide sequence that isoperatively linked with another polynucleotide sequence that encodes theamino acid sequence of a polypeptide chain to effect the expression ofthat encoded amino acid sequence. The regulatory sequence can inhibit,repress, promote, or even drive the expression of the operably-linkedpolynucleotide sequence encoding the amino acid sequence.

The term “selectable marker” or “selective marker” refers to apolynucleotide (e.g., a gene) capable of expression in a host cell,which allows for ease of selection of those hosts containing the vector.Examples of selectable markers include but are not limited toantimicrobial markers. Thus, the term “selectable marker” refers to agene that provides an indication when a host cell has taken up anincoming sequence of interest or when some other reaction has takenplace. Typically, selectable markers are genes that confer antimicrobialresistance or a metabolic advantage on the host cells to allow the cellscontaining the exogenous sequences to be distinguished from the cellsthat have not received the exogenous sequences. A “residing selectablemarker” is one that is located on the chromosome of the microorganism tobe transformed. A residing selectable marker encodes a gene that isdifferent from the selectable marker on the transforming construct.Selective markers are known to those of skill in the art. As indicatedabove, suitably the marker is an antimicrobial resistant marker,including, for example, amp^(R); phleo^(R); spec^(R); kan^(R); ery^(R);tet^(R); cmp^(R); and neo^(R). See, e.g., Guerot-Fleury, Gene,167:335-337, 1995; Palmeros et al., Gene, 247:255-264, 2000; andTrieu-Cuot et al., Gene, 23:331-341, 1983. Other markers useful inaccordance with the invention include, but are not limited to,auxotrophic markers, such as tryptophan; and detection markers, such as6-galactosidase.

The term “selectable marker-encoding nucleotide sequence” refers to apolynucleotide sequence that is capable of expression in the host cellsand where the expression of the selectable marker confers to the cellscontaining the expressed gene the ability to grow in the presence of acorresponding selective agent or in the absence of one or more essentialnutrients.

A “signal sequence” or “signal peptide” refers to a polynucleotide oramino acid sequence that participates in the secretion of the mature orprecursor forms of a protein. This definition of signal sequence is afunctional one, meant to include all those amino acid sequences encodedby the N-terminal portion of the protein gene, which participate in theeffectuation of the secretion of protein. They are often, but notuniversally, bound to the N-terminal portion of a protein or to theN-terminal portion of a precursor protein. The signal sequence can beendogenous or exogenous. The signal sequence can be one that is normallyassociated with the protein (e.g., thioesterase), or can be oneoriginated or derived from a gene encoding another secreted protein. Anexemplary exogenous signal sequence comprises the first seven amino acidresidues of the signal sequence from Bacillus subtilis subtilisin fusedto the remainder of the signal sequence of the subtilisin from Bacilluslentus (ATCC 21536). Another exemplary signal sequence comprises thesignal sequence for TesA that is removed to produce 'TesA.

The term “substantially identical,” in the context of twopolynucleotides or two polypeptides refers to a polynucleotide orpolypeptide that comprises at least 70% sequence identity, for example,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity as comparedto a reference sequence using the programs or algorithms (e.g., BLAST,ALIGN, CLUSTAL) using standard parameters. One indication that twopolypeptides are substantially identical can be that the firstpolypeptide is immunologically cross-reactive with the secondpolypeptide. Typically, polypeptides that differ by conservative aminoacid substitutions are immunologically cross-reactive. Thus, apolypeptide is substantially identical to a second polypeptide, forexample, when the two peptides differ only by a conservativesubstitution. Another indication that two polynucleotide sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions (e.g., within a range of medium tomaximum stringency).

“Substantially purified” means molecules that are at least about 60%free, preferably at least about 75% free, about 80% free, about 85%free, and more preferably at least about 90% free from other componentswith which they are naturally associated. As used herein, the term“purified” or “to purify” also refers to the removal of contaminantsfrom a sample. For example, the removal of contaminants can result in anincrease in the percentage of fatty acid derivatives of interest in asample. For example, after fatty acid derivatives are expressed inplant, bacterial, yeast, or mammalian production host cells, the fattyacid derivatives can be purified by, e.g., the removal of productionhost cell proteins. This step, also called recovery, involves separatingand processing the fatty acid derivative composition such that thecomposition is useful in industrial applications, for example, as a fuelor a chemical. After purification, the percentage of fatty acidderivatives in the sample is increased. The term purified does notrequire absolute purity; rather, it is intended as a relative term.Thus, for example, a purified fatty acid derivative preparation is onein which the product is more concentrated than the product is in itsenvironment within a cell. For example, a purified fatty ester is onethat is substantially separated from cellular components (e.g.,polynucleotides, lipids, carbohydrates, and other peptides) that canaccompany it. In another example, a purified fatty ester preparation isone in which the fatty ester is substantially free from contaminants,such as those that might be present following fermentation. For example,a fatty ester is said to be “purified” when at least about 50% by weightof a sample is composed of the fatty ester. In another example when atleast about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more by weight of a sample is composed of the fattyester.

“Substitution” means replacing an amino acid in the sequence of aprecursor protein with another amino acid at a particular position,resulting in a mutant of the precursor protein. The amino acid used as asubstitute can be a naturally-occurring amino acid, or can be asynthetic or non naturally-occurring amino acid.

The term “surfactants” refers to substances that are capable of reducingthe surface tension of a liquid in which they are dissolved. They aretypically composed of a water-soluble head and a hydrocarbon chain ortail. The water-soluble head is hydrophilic and can be either ionic ornonionic. The hydrocarbon chain is hydrophobic. Surfactants are used ina variety of products, including detergents and cleaners, and are alsoused as auxiliaries for textiles, leather and paper, in chemicalprocesses, in cosmetics and pharmaceuticals, in the food industry, inagriculture, and in oil recovery. In addition, they can be used to aidin the extraction and isolation of crude oils which are found inhard-to-access environments or in water emulsions. There are four typesof surfactants characterized by varying uses. Anionic surfactants havedetergent-like activity and are generally used for cleaningapplications. Cationic surfactants contain long chain hydrocarbons andare often used to treat proteins and synthetic polymers or arecomponents of fabric softeners and hair conditioners. Amphotericsurfactants also contain long chain hydrocarbons and are typically usedin shampoos. Non-ionic surfactants are often used in cleaning products.

The term “synthase” refers to an enzyme that catalyzes a synthesisprocess. As used herein, the term “synthase” includes synthases andsynthetases.

The term “target property” refers to a property of the starting genethat is intended to be altered.

The term “thioesterase” refers to an enzyme that has thioesteraseactivity. Thioesterases include thioester hydrolases, which areidentified as members of Enzyme Classification E.C. 3.1.2 and areobtainable from a variety of sources. Plant thioesterases are describedin, for example, Voelker and Davies, J. Bact., Vol., 176, No. 23, pp.7320-27, 1994, U.S. Pat. Nos. 5,667,997, and 5,455,167. Thioesterasesare also obtainable from microbial sources, such as those described inAkoh et al., Prog. Lipid Res., vol. 43, no. 6, pp. 534-52, 2004;Diczfalusy and Alexson, Arch. Biochem. Biophys., vol. 334, no. 1, pp.104-12, 1996; Larson and Kolattukudy, Arch. Biochem. Biophys., vol. 237,no. 1, pp. 27-37, 1985; Lawson et al., Biochemistry, vol. 33, no. 32,pp. 9382-88, 1994; Lee et al., Eur. J. Biochem., vol. 184, no. 1, pp.21-28, 1989; Naggert et al., J. Biol. Chem., vol. 266, no. 17, pp.11044-50, 1991; Nie et al., Biochemistry, vol. 47, no. 29, pp. 7744-51,2008; Seay and Lueking, Biochemistry, vol. 25, no. 9, pp. 2480-85, 1986;Spencer et al., J. Biol. Chem., vol. 253, no. 17, pp. 5922-26, 1978; andZhuang et al., Biochemistry, vol. 47, no. 9, pp. 2789-96, 2008.Thioesterases are also obtainable from, for example, cyanobacterial,algal, mammalian, insect, and fungal sources. A thioesterase can haveactivity other than thioesterase activity, for example proteolyticactivity or oxygen ester hydrolysis activity. A particularly usefulthioesterase is the 'TesA (or thioesterase I) enzyme from E. coli, whichis a truncated version of the full-length TesA serine thioesteraseenzyme that is described in Cho and Cronan, J. Biol. Chem., vol., 268,no. 13, pp. 9238-45, 1992. An E. coli 'TesA polypeptide comprises 182amino acids, and is the product of a cleavage reaction wherein the 26amino acid leader sequence of E. coli TesA is removed. E. coli 'Tes A,for example, has the amino acid sequence of SEQ ID NO:31 in FIG. 58.

The term “thioesterase activity” refers to the capacity to catalyze athioester cleavage reaction, which usually involves the hydrolysis of athioester at a thiol group into an acid and a thiol, but can alsoinclude a transesterification step in which a thioester bond is cleavedand a new ester bond is formed. In general, an acyl-ACP thioesterase iscapable of catalyzing the hydrolytic cleavage of fatty acyl-acyl carrierprotein thioesters and/or fatty acyl-coenzyme A thioesters. Examples ofenzymes having thioesterase activity include acetyl-CoA hydrolase,palmitoyl-CoA hydrolase, succinyl-CoA hydrolase, formyl-CoA hydrolase,acyl-CoA hydrolase, palmitoyl-protein thioesterase, and ubiquitinthiolesterase. Thioesterase activity can be established by any of thefollowing assays:

Acyl-CoA Hydrolysis Assay:

-   -   A Tris-HCl buffer, 0.1 M, pH 8.0; Palmitoyl-CoA, 5 μM; DTNB,        0.01 M in 0.1 M potassium phosphate buffer, pH 7.0 are used to        prepare a complete assay mixture. The assay mixture thus        contains a final concentration of 10 μmol of Tris-HCl buffer, pH        8.0, 0.05 μmol of DTNB, and 0.01 μmol of palmitoyl-CoA. The        complete assay mixture is then mixed with the thioesterase, in a        final volume of 2.0 mL.    -   The rate of cleavage of the acyl-CoA substrate is measured by        monitoring the change in absorbance at 405 nm, using a molar        extinction coefficient of 13,600 M⁻¹ cm⁻¹.

In Vivo Assay:

-   -   The thioesterase of interest is expressed in a suitable host,        such as an E. coli. Following expression of the protein, the        culture is acidified with 1 N HCl to a final pH of about 2.5 and        then extracted with an equal volume of ethyl acetate. Free fatty        acids in the organic phase are derivatized with        tetramethylammonium hydroxide (TMAH) to generate the respective        methyl esters, which are then analyzed on a gas chromatograph        equipped with a flame ionization detector.

Thiolactone Hydrolysis Assay:

-   -   A reagent solution containing 25 mM L-homocysteine thiolactone        (L-HcyT) and 0.5 mM 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) in        0.1 M HEPES buffer (pH 7.3) is first prepared. Enzyme is then        added to the reagent solution and L-HcyT hydrolysis is monitored        by detecting the free thiol group with DTNB at 412 nm (c=13,600        M⁻¹ cm⁻¹ for 5-thio-2-nitrobenzoic acid). 4-MU-6S-Palm-βGlc        Assay:    -   A reaction mixture containing 10 μL of thioesterase enzyme and        20 μL of substrate solution is first prepared. The substrate        solution contains 0.64 mM MU-6S-Palm-I3-Glc, 15 mM        dithiothreitol (DTT), 0.375% (w/v) Triton X-100, and 0.1 U        β-glucosidase from almonds in McIlvain's phosphate/citrate        buffer, pH 4.0. The reaction mixture is incubated for 1 hour at        37° C. Exogenous almond β-glucosidase is added to hydrolyze the        reaction intermediate, MU-6-thio-β-glucoside, quantitatively.        The hydrolysis reaction is terminated by the addition of 200 μL        of 0.5 M sodium carbonate, pH 10.7, containing 0.025% Triton        X-100, and the fluorescence of the released        4-methylumbelliferone (MU) is measured in a fluorometer        (λ_(ex)=372, λ_(em)=445 nm).

Lysophospholipase Assay:

-   -   A reaction mixture containing 10 μL of thioesterase mixed with        10 μL of 3 mM 1-oleoyl-phosphatidylethanolamine, 25 μL of 100 mM        Tris-HCl (pH 7.0), and 5 μL of 5 mM EDTA is prepared. The        reaction is terminated with the addition of 1.5 mL CHCl₃:CH₃OH        (1:2), followed by the addition of water to bring the total        aqueous volume to 0.9 mL. The organic phase is then analyzed by        thin layer chromatography together with suitable standards,        using plates prepared from 40 g Silica Gel H suspended in 95 mL        of 1 mM sodium tetraborate. The solvent system consists of        CHCl₃:CH₃OH:H₂O (95:35:5).

Protease Substrate Assay:

-   -   A reaction mixture containing 10 μL of enzyme mixed with 800 μL        12.5 mM Tris-HCl (pH 8.0) containing 0.25% Triton X-100 and 10        μL of Cbz-Phe-ONp dissolved in DMSO is prepared. The        p-nitrophenol released via cleavage of the substrate is measured        by monitoring the absorbance at 405 nm.

Fatty Acyl-PNP Hydrolysis Assay:

-   -   A reagent solution containing 2% Triton X-100 in 50 mM sodium        phosphate, pH 7.0, and 10 mM C₁₂-p-nitrophenol (acyl-PNP) in        acetone is first prepared. Then a C₁₂-PNP working solution is        prepared by mixing 600 μL 10 mM C₁₂-PNP into a 9.4-mL phosphate        buffer.    -   The assay is performed by adding 40 μL of the acyl-PNP working        solution to each well of a 96-well plate, followed by the rapid        addition of 40 μL of enzyme. The solution is mixed for 15        seconds, and the absorbance change is read at 405 nm in a        microtiter plate reader at 25° C.

Ester Formation from Thioester:

-   -   A reaction mixture containing 1.5 μM thioesterase enzyme, 100 μM        myristoyl-CoA, 10% (v/v) methanol, and 50 mM sodium phosphate,        pH 7.0 is prepared. The reaction mixture is incubated for 1 hour        at 20° C. and terminated with the addition of 1 N HCl to        decrease the pH to about 2.5. The mixture is extracted with an        equal volume of ethyl acetate and the amount of fatty ester        produced is determined via GC-MS or other standard methods such        as GC-FID, LC-MS, or thin layer chromatography.

Ester Formation from Ester:

-   -   A reaction mixture containing 1.5 μM thioesterase enzyme, 300 μM        lauroyl-CoA, 10% (v/v) methanol, and 50 mM sodium phosphate, pH        7.0 is prepared. The reaction mixture is incubated for 1 hour at        20° C. and terminated with the addition of 1 N HCl to decrease        the pH to about 2.5. The mixture is extracted with an equal        volume of ethyl acetate and the amount of lauryl ester produced        is determined via GC-MS or other standard methods such as        GC-FID, LC-MS, or thin layer chromatography.

The term “transformed” or “stably transformed” cell refers to a cellthat has a non-native (heterologous) polynucleotide sequence integratedinto its genome or as an episomal plasmid that is maintained for atleast two generations.

The term “transport protein” refers to a protein that facilitates themovement of one or more compounds in and/or out of an organism ororganelle. In some embodiments, an exogenous DNA sequence encoding anATP-Binding Cassette (ABC) transport protein will be functionallyexpressed by the production host so that the production host exports thefatty acid derivative into the culture medium. ABC transport proteinsare found in many organisms, such as Caenorhabditis elegans, Arabidopsisthalania, Alcaligenes eutrophus (later renamed Ralstonia eutropha), orRhodococcus erythropolis. Non-limiting examples of ABC transportproteins include CER5, AtMRP5, AmiS2 and AtPGP1. In a preferredembodiment, the ABC transport protein is CER5 (e.g., AY734542). In otherembodiments, the transport protein is an efflux protein selected from:AcrAB, To1C, or AcrEF from E. coli or t111618, t111619, and t110139 fromThermosynechococcus elongatus BP-1. In further embodiments, thetransport protein is a fatty acid transport protein (FATP) selected fromDrosophila melanogaster, Caenorhabditis elegans, Mycobacteriumtuberculosis, or Saccharomyces cerevisiae or any one of the mammalianFATPs known in the art. Transport proteins are useful, for example, forenhancing the secretion or release of products that are otherwise notcapable of spontaneously secret the product. They are also useful whenthe engineered host cells are capable of spontaneously secret or releasethe product, but either release it slowly or incompletely. Under thosecircumstances, the transport proteins can enhance the secretion byaccelerating the secretion step or driving the secretion to completion.

“Variant” is used interchangeably herein with “mutant.”

“Vector” refers to a polynucleotide construct designed to introducepolynucleotides into one or more cell types. Vectors include cloningvectors, expression vectors, shuttle vectors, plasmids, cassettes andthe like. In some embodiments, the polynucleotide construct comprises apolynucleotide sequence encoding a thioesterase (e.g., a precursor or amature thioesterase) that is operably linked to a suitable prosequence(e.g., a secretory pro-sequence) capable of effecting the expression ofthe polynucleotide or gene in a suitable host.

A “wax” is a substance comprising, at least in part, fatty esters. Incertain embodiments, a fatty ester has an A side and a B side, eachcomprising medium to long carbon chains. In addition to fatty esters, awax may comprise other components. For example, a wax can comprisehydrocarbons, sterol esters, aliphatic aldehydes, alcohols, ketones,beta-diketones, triacylglycerols and the like. Typically a wax is asolid at room temperature, for example, at 20° C.

“Wild-type” means, in the context of gene or protein, a polynucleotideor protein sequence that occurs in nature. In some embodiments, thewild-type sequence refers to a sequence of interest that is a startingpoint for protein engineering.

Production of Fatty Acid Derivatives

According to an embodiment of the present invention, the novelthioesterases of the invention are expressed in a host cell that iscapable of converting a carbon source to a fatty acid derivative. Theinvention pertains to two distinct embodiments: (1) the discovery that amutant thioesterase can be used to optimize and/or “design” a fatty acidderivative composition so as to make such compositions more useful andthat different mutations will provide different target properties; and(2) the discovery that thioesterase will act in a recombinant host cellto directly produce fatty ester products, without the presence of a waxsynthase or ester synthase enzyme.

According to an embodiment of the invention, the fatty acid derivativecompositions produced in accordance with the methods, vectors, and cellsherein have modified or altered properties as compared to the fatty acidderivatives produced using host cells that do not comprise thethioesterase variants of the invention. For example, as also describedherein, using the thioesterases of the present invention, it is possibleto develop manufacturing processes that produce fatty acid derivatives,which, in comparison with a similar process involving a wildtypethioesterase, have altered compositional profiles, for example, alteredpercentages of a range of or a specific carbon chain length acyl group,saturated or unsaturated acyl groups, position of unsaturations,branched acyl groups, position of branching, hydroxyl-acyl groups,keto-acyl groups, proportion of esters or free fatty acids in theproduct, proportion of short-chain (e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃,and/or C₁₄) vs. long-chain (e.g., C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, and/or C₂₀)fatty acid derivatives, or yield of fatty acid derivatives. Accordingly,products with various desirable properties can be engineered such thatthey have optimized cetane numbers, octane ratings, oxidative stability,lubricity, flash points, viscosity, boiling points, melting points, pourpoints, cloud points, cold filter plugging points, cold flowcharacteristics, aromaticity, and/or iodine numbers.

Fatty acid derivatives are useful as, or as components of, biofuels andspecialty chemicals. Fatty acid derivatives and products made therefrominclude fuels, fuel additives, fuel blends, detergents and surfactants,nutritional supplements, polymers, paraffin replacements, lubricants,solvents, personal care products, rubber processing additives, corrosioninhibitors, emulsifiers, plastics, textiles, cosmetics, paper products,coatings, metalworking fluids, dielectrics, oiling agents andemollients. The methods and compositions disclosed herein allow for theproduction of fatty acid derivatives with particular branch points,levels of saturation, and carbon chain length. The methods andcompositions herein also allow for the production of a higher proportionof fatty esters vs. other products, or alternatively, a lower proportionof fatty esters vs. other products, depending on whether a higherproportional or percentage yield of fatty esters or a lower proportionalor percentage yield of fatty esters is desirable. Specifically, forexample, the methods and compositions herein allow for the production ofa larger proportion of fatty acid esters vs. free fatty acids, or inother words, allows for a higher proportional or percentage yield offatty acid esters vs. free fatty acids. Alternatively, for example, themethods and compositions herein allow for the production of a smallerproportion of fatty acid esters vs. free fatty acids, when large amountsof fatty acid esters are undesirable. Furthermore, the methods andcompositions herein allow for the production of an improved yield offatty acid derivatives.

Non-limiting examples of microorganisms which can be used as productionhosts to produce fatty acid derivatives include cyanobacteria, algae,bacteria, yeast, or filamentous fungi. Further non-limiting examples ofsuitable production hosts include plant, animal, or human cells.

Alcohols (short chain, long chain, branched, or unsaturated) can beproduced by the production hosts described herein. Such alcohols can beused as fuels directly or they can be used to create a fatty ester.Fatty esters, alone or in combination with other fatty acid derivativesdescribed herein, are also useful as, or as components of, fuels.

Similarly, hydrocarbons produced from the production hosts describedherein can be used as, or as components of, biofuels. Suchhydrocarbon-based fuels can be designed to contain branch points,defined degrees of saturation, and specific carbon lengths utilizing theteachings provided herein. When used as biofuels alone or in combinationwith other fatty acid derivatives, the hydrocarbons can be combined withsuitable additives or other traditional fuels (e.g., alcohols, dieselderived from triglycerides, and petroleum-based fuels).

The cetane number (CN), viscosity, melting point, and heat of combustionfor various fatty esters have been characterized in Knothe, FuelProcessing Technology 86:1059-1070, 2005, which is herein incorporatedby reference in its entirety. A production host can be engineered toproduce any of the fatty esters described in Knothe, using the teachingsprovided herein.

I. Production of Fatty Acid Derivatives and Modifications for ImprovingProduction/Yield

The production host used to produce acyl-CoA and/or fatty acidderivatives can be recombinantly modified to include polynucleotidesequences that over-express peptides. For example, the production hostcan be modified to increase the production of acyl-CoA and reduce thecatabolism of fatty acid derivatives and intermediates in the fatty acidbiosynthetic pathway, or to reduce feedback inhibition at specificpoints in the fatty acid biosynthetic pathway. In addition to modifyingthe genes described herein, additional cellular resources can bediverted to over-produce fatty acids. For example, the lactate,succinate, and/or acetate pathways can be attenuated, and acetyl-CoAcarboxylase (acc) can be over-expressed. The modifications to theproduction host described herein can be through genomic alterations,addition of recombinant expression systems, or combinations thereof. Forexample, one or more endogenous thioesterases of a particular productionhost can be modified using suitable techniques such that the mutantthioester has at least one altered property as compared to theendogenous thioesterase precursor, or such that the host cell exhibitsat least one altered property, as compared to the same host cell beforeit is subject to the genomic alteration steps.

The fatty acid biosynthetic pathways involved are illustrated in FIGS.2-5. Subsections A-G below describe the steps in these pathways. Variousenzymes catalyze various steps in the pathway. Accordingly, each step isa potential place for overexpression of the gene to produce moreenzyme(s) and thus drive the production of more fatty acids and fattyacid derivatives. Genes encoding the enzymes required for the pathwaymay also be recombinantly added to a production host lacking suchenzymes. Finally, steps that would compete with the pathway leading toproduction of fatty acids and fatty acid derivatives can be attenuatedor blocked in order to increase the production of the desired products.

According to the disclosures herein, a person of ordinary skill in theart can use the thioesterases of the invention to prepare microorganismsthat produce fatty acid derivatives and to manufacture various fattyacid derivatives using such microorganisms, wherein such fatty acidderivatives have altered properties. It is further possible to preparemicroorganisms that produce such fatty acid derivatives more efficientlyby having the desired levels of yield, productivity, or titer duringfermentations.

A. Acetyl-CoA—Malonyl-CoA to Acyl-ACP

Fatty acid synthase (FAS) is a group of peptides that catalyze theinitiation and elongation of acyl chains (Marrakchi et al., BiochemicalSociety, 30:1050-1055, 2002). The acyl carrier protein (ACP) along withthe enzymes in the FAS pathway control the length, degree of saturation,and branching of the fatty acids produced. The steps in this pathway arecatalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoAcarboxylase (acc) gene families. Depending upon the desired product, oneor more of these genes can be attenuated or over-expressed.

I. Fatty Acid Biosynthetic Pathway: Acetyl-CoA or Malonyl-CoA toAcyl-ACP

The fatty acid biosynthetic pathway in the production host uses theprecursors acetyl-CoA and malonyl-CoA. The steps in this pathway arecatalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoAcarboxylase (acc) gene families. This pathway is described in Heath etal., Prog. Lipid Res., 40(6):467-97, 2001, which is incorporated hereinby reference.

Acetyl-CoA is carboxylated by acetyl-CoA carboxylase (Acc, amulti-subunit enzyme encoded by four separate genes, accABCD) to formmalonyl-CoA. The malonate group is transferred to ACP by malonyl-CoA:ACPtransacylase (FabD) to form malonyl-ACP. A condensation reaction thenoccurs, where malonyl-ACP merges with acetyl-CoA, resulting inβ-ketoacyl-ACP. β-ketoacyl-ACP synthase III (FabH) initiates the FAScycle, while β-ketoacyl-ACP synthase I (FabB) and β-ketoacyl-ACPsynthase II (FabF) are involved in subsequent cycles.

Next, a cycle of steps is repeated until a saturated fatty acid of theappropriate length is made. First, the β-ketoacyl-ACP is reduced byNADPH to form β-hydroxyacyl-ACP. This step is catalyzed byβ-ketoacyl-ACP reductase (FabG). β-hydroxyacyl-ACP is then dehydrated toform trans-2-enoyl-ACP. β-hydroxyacyl-ACP dehydratase/isomerase (FabA)or β-hydroxyacyl-ACP dehydratase (FabZ) catalyze this step.NADPH-dependent trans-2-enoyl-ACP reductase I, II, or III (Fabl, FabK,or FabL, respectively) reduces trans-2-enoyl-ACP to form acyl-ACP.Subsequent cycles are started by the condensation of malonyl-ACP withacyl-ACP by β-ketoacyl-ACP synthase I or β-ketoacyl-ACP synthase II(FabB or FabF, respectively).

II. Modifying the Fatty Acid Biosynthetic Pathway to Increase Acyl-ACPProduction

Production host organisms may be engineered to overproduce acetyl-CoAand malonyl-CoA. Such production host organisms include plant, animal,or human cells. Microorganisms such as cyanobacteria, algae, bacteria,yeast, or filamentous fungi can be used as production hosts.Non-limiting examples of microorganisms that may be used as productionhosts include E. coli, Saccharomyces cerevisiae, Candida lipolytica,Synechococcus, Synechocystis, Clamydomonas, Arthrobacter AK 19,Rhodotorula glutinins, Acinetobacter sp. strain M-1, Candida lipolytica,and other oleaginous microorganisms. Several different modifications canbe made, either in combination or individually, to the production hostto obtain increased acetyl-CoA/malonyl-CoA/fatty acid and fatty acidderivative production.

For example, to increase acetyl-CoA production, one or more of thefollowing genes can be expressed in a production host: pdh, panK, aceEF(which encodes the E1p dehydrogenase component and the E2pdihydrolipoamide acyltransferase component of the pyruvate and2-oxoglutarate dehydrogenase complexes), fabH, fabD, fabG, acpP, andfabF. In other examples, additional genes encoding fatty-acyl-CoAreductases and aldehyde decarbonylases can be expressed in theproduction host. It is known in the art that a plasmid containing one ormore of the aforementioned genes, all under the control of aconstitutive, or otherwise controllable promoter, can be constructed.Exemplary GenBank Accession numbers for these genes are listed in theparentheticals: pdh (BAB34380, AAC73227, AAC73226), panK (also known ascoaA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175), fabD(AAC74176), fabG (AAC74177), acpP (AAC74178), and fabF (AAC74179).

Additionally, the expression levels of fadE, gpsA, ldhA, pflb, adhE,pta, poxB, ackA, and/or ackB can be attenuated or knocked-out in theengineered microorganism by transformation with conditionallyreplicative or non-replicative plasmids containing null or deletionmutations of the corresponding genes, or by substituting the promoter orenhancer sequences. Exemplary GenBank Accession numbers for these genesare listed in the parentheticals: fadE (AAC73325), gspA (AAC76632), ldhA(AAC74462), pflb (AAC73989), adhE (AAC74323), pta (AAC75357), poxB(AAC73958), ackA (AAC75356), and ackB (BAB81430). The resultingengineered production hosts have increased acetyl-CoA production levelswhen grown in an appropriate environment.

Moreover, malonyl-CoA overproduction can be affected by engineering theproduction host as described above with accABCD (e.g., GenBank Accessionnumber AAC73296, EC 6.4.1.2) included in the plasmid synthesized denovo. Fatty acid overproduction can be achieved by further including agene encoding lipase (e.g., GenBank Accession Nos. CAA89087 andCAA98876) in the plasmid synthesized de novo.

As a result, in some examples, an acetyl-CoA carboxylase isoverexpressed to increase the intracellular concentration thereof by atleast about 2-fold, at least about 5-fold, or at least about 10-fold,relative to the native expression levels.

In addition, a PlsB (e.g., GenBank Accession number AAC77011) D311Emutant can be used to increase the amount of available acyl-CoA.

In addition, overexpression of an sfa gene (suppressor of FabA, e.g.,GenBank Accession No. AAN79592) can be included in the production hostto increase production of monounsaturated fatty acids (Rock et al., J.Bacteriology, 178:5382-5387, 1996).

B. Acyl-ACP and/or Acyl-CoA to Fatty Ester Using Thioesterase

In a typical microbial process model for fatty acid synthesis,acetyl-CoA and malonyl-CoA are converted through a series of steps toform the acyl-ACP chains. Acyl-ACP is then converted via a series ofalternative enzymatic steps to various end products, including fattyacid derivatives. For example, typically acyl-ACP is converted to fattyesters by the combined consecutive reactions of a thioesterase, anacyl-CoA ligase/synthetase and an ester synthase. A limitation to thecommercial use of these enzymes in a metabolic pathway is the need toproduce the fatty acyl CoA substrate from a fatty acyl ACP precursor,which requires at least two enzymatic steps and the expenditure ofmetabolic energy from two phosphoanhydride bonds. Direct production offatty esters with thioesterase mitigates the loss of ATP caused by thesetwo enzymatic steps. Recently it has been demonstrated that lipases(whose natural “alcohol” substrate is water) can also be used in vitroto catalyze the transesterification reaction that makes biodiesel (i.e.the conversion of triacyl glyceride and methanol to fatty acid methylester and glycerol). However, lipases are generally toxic to the cellswhen produced intracellularly.

Despite having a published specificity for water, the present inventiondescribes the discovery that, in the presence of a sufficient amount ofan alcohol, the alcohol can become an acceptable substrate for athioesterase. In that case, thioesterases can catalyze the alcoholysisof the fatty acyl enzyme intermediates, just like a lipase does invitro. Thus, under the right conditions, an enzyme that accepts a fattyester as substrate to form a fatty enzyme intermediate that issubsequently cleaved through either hydrolysis or transesterificationcan be used to synthesize desired fatty acid esters if a sufficientlevel of a suitable alcohol is provided to drive alcoholysis. Examplesof enzymes having this capability, which can produce esters directlyfrom acyl-ACP include, in addition to thioesterases, acyltransferases,lipases, esterases, and proteases. Useful thioesterases can benaturally-occurring and/or precursor thioesterases as defined herein, orcan be mutant thioesterases prepared in accordance with the disclosuresherein. One of ordinary skill in the art is capable of determining thefitness of using a particular enzyme to directly produce fatty estersfrom Acyl-ACP. For example, the assays provided in Example 32 are usefulin determining direct ester production.

According to this aspect of the invention, the thioesterase can beutilized to directly produce fatty esters either in the presence or theabsence of an ester synthase and/or a fatty acyl CoA ligase/synthetase.For example, expression of a thioesterase that can catalyze the directproduction of fatty esters in a recombinant host strain can be used tosupplement fatty ester production where the strain also expresses anester synthase. Additionally, expression of a thioesterase that cancatalyze the direct production of fatty esters in a recombinant hostcell can be used where there is no or low ester synthase expression.

A mutant thioesterase can be utilized that has been modified to havealtered properties compared to the precursor thioesterase.

C. Acyl-ACP to Fatty Acid

I. Fatty Acid Biosynthetic Pathway: Acyl-ACP to Fatty Acids

As described above, acetyl-CoA and malonyl-CoA are processed in severalsteps to form acyl-ACP chains. The enzyme sn-glycerol-3-phosphateacyltransferase (PlsB) catalyzes the transfer of an acyl group fromacyl-ACP or acyl-CoA to the sn-1 position of glycerol-3-phosphate. Thus,PlsB is a key regulatory enzyme in phospholipid synthesis, which is apart of the fatty acid pathway. Inhibiting PlsB leads to an increase inthe levels of long chain acyl-ACP, which feedback will inhibit earlysteps in the pathway, which involve genes such as, for example, accABCD,fabH, and fabI. Uncoupling of this regulation, for example bythioesterase overexpression, leads to increased fatty acid production.

II. Modifying the Fatty Acid Biosynthetic Pathway to Produce the DesiredTypes or Proportions of Fatty Acids

According to the invention, the expressed thioesterase has alteredproperties as compared to the native or endogenous thioesterase in thehost strain. To engineer a production host for the production of ahomogeneous population of fatty acid derivatives, one or more endogenousgenes can be attenuated or functionally deleted and, as a result, one ormore thioesterases according to the invention can be expressed. Forexample, C₁₀ fatty acid derivatives (i.e., fatty acid derivatives eachcomprising a carbon chain that is 10 carbons long) can be produced byattenuating thioesterase C₁₈ (e.g., GenBank Accession Nos. AAC73596 andPOADA1), which uses C_(18:1)-ACP, and by expressing an alteredthioesterase that has increased specificity for and/or activity (e.g.,catalytic rate) with regard to C₁₀ substrates (i.e., substrates eachcomprising a carbon chain that is 10 carbons long). This results in amore homogeneous population of fatty acid derivatives that have anincrease in fatty acids having a carbon chain length of 10. In anotherexample, C₁₂ fatty acid derivatives can be produced by attenuatingendogenous thioesterases that produce non-C₁₂ fatty acids and expressingan altered thioesterase that has increased specificity for and/oractivity (i.e., catalytic rate) with regard to C₁₂ substrates. Inanother example, C₁₄ fatty acid derivatives can be produced byattenuating endogenous thioesterases that produce non-C₁₄ fatty acidsand expressing an altered thioesterase that has increased specificityfor and/or activity (i.e., catalytic rate) with regard to C₁₄substrates. In another example, a higher proportional yield ofshort-chain (e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, and/or C₁₄) fatty acidderivatives vs. other non-short-chain fatty acid derivatives in theproduct mixture. In yet another example, a lower proportional yield ofshort chain (e.g., C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, and/or C₁₄) fatty acidderivatives vs. other non-short-chain fatty acid derivatives in theproduct mixture can also be achieved. In a further example, a higherand/or improved yield of free fatty acid derivatives can be produced byexpressing an altered thioesterase that has improved catalytic rateand/or production or yield in vivo. In yet another example, a higher orlower proportional or percentage yield of fatty esters vs. otherproducts, such as free fatty acids, can be produced by applying one ormore of certain thioesterase mutants. Acetyl-CoA, malonyl-CoA, and fattyacid overproduction can be verified using methods known in the art, forexample by radioactive precursors, HPLC, LC-MS, and GC-MS subsequent tocell lysis.

In an alternative embodiment, a thioesterase of the invention can beexpressed within the host strain in combination with an endogenousthioesterase. In yet another alternative embodiment, one or moreendogenous thioesterases can be modified using suitable genomicalternation techniques that are known to those skilled in the art, suchthat the mutant thioesterases has at least one altered property ascompared to the endogenous thioesterase precursors, and/or such that thehost cell exhibits at least one altered property as compared to the hostcell before such genomic alteration techniques are applied.

D. Fatty Acid to Acyl-CoA

I. Conversion of Fatty Acids to Acyl-CoA

Acyl-CoA synthase (ACS) esterifies free fatty acids to acyl-CoA by atwo-step mechanism. The free fatty acid first is converted to anacyl-AMP intermediate (an adenylate) through the pyrophosphorolysis ofATP. The activated carbonyl carbon of the adenylate is then coupled tothe thiol group of CoA, releasing AMP and the acyl-CoA final product.See Shockey et al., Plant Physiol. 129:1710-1722, 2002.

The E. coli ACS enzyme FadD and the fatty acid transport protein FadLare typically important components of a fatty acid uptake system. FadLmediates the transportation of fatty acids into the bacterial cell, andFadD mediates the formation of acyl-CoA esters. When no other carbonsource is available, exogenous fatty acids are taken up by bacteria andconverted to acyl-CoA esters, which bind to the transcription factorFadR and derepress the expression of the fad genes that encode proteinsresponsible for fatty acid transport (FadL), activation (FadD), andβ-oxidation (FadA, FadB, FadE, and FadH). When alternative sources ofcarbon are available, bacteria synthesize fatty acids as acyl-ACPs,which are then used for phospholipid synthesis, rather than serving assubstrates for β-oxidation. Thus, acyl-CoA and acyl-ACP are independentsources of fatty acids that lead to different end-products. See Cavigliaet al., J. Biol. Chem., 279(12):1163-1169, 2004.

II. Modifying the Fatty Acid Biosynthetic Pathway to Increase Conversionof Fatty Acids to Acyl-CoA

Production hosts can be engineered using known peptides to produce fattyacids of various lengths which can be converted to acyl-CoA. One methodof making fatty acid derivatives involves increasing the expression of,or expressing more active forms of, one or more acyl-CoA synthasepeptides (EC 6.2.1.−).

A list of acyl-CoA synthases that can be expressed to produce acyl-CoAand fatty acid derivatives is shown in Table 1. These Acyl-CoA synthasescan be examined to optimize any pathway that uses fatty-acyl-COAs assubstrates. Using bioinformatics and synthetic genes, heterologous fadDgenes can be expressed in production strains and evaluated for theircapacity to produce biodiesel and potentially biocrude.

TABLE 1 Acyl-CoA synthases % Similarity Gene GenBank % Identity to to E.coli Name/Locus Source Accession No. E. coli FadD FadD fadD E. coliNP_416319 — — fadK E. coli YP_416216 28 46 fadD Acinetobacter sp. ADP1YP_045024 51 70 fadD Haemophilus influenza RdKW20 NP_438551 64 78 BH3103Bacillus halodurans C-125 NP_243969 40 58 yhfL Bacillus subtilisNP_388908 39 57 pfl-4354 Pseudomonas fluorescens Pfo-1 YP_350082 52 71EAV15023 Comamonas testosterone KF-1 ZP_01520072 55 72 fadD1 Pseudomonasaeruginosa NP_251989 54 72 fadD2 Pseudomonas aeruginosa PAO1 NP_25199055 72 fadD Rhizobium etli CFN42 YP_533919 55 72 RPC_4074Rhodopseudomonas palustris Bis B18 YP_533919 56 72 fadD1 Rasltoniasolanacearum GMI 1000 NP_520978 56 72 fadDD35 Mycobacterium tuberculosisH37Rv NP_217021 28 46 fadDD22 Mycobacterium tuberculosis H37Rv NP_21746423 42 PRK0059 Stenotrophomonas maltophilia R551-3 ZP_01644857 59 75

Based on their degree of similarity to E. coli fadD, the followinghomologous genes are selected to be synthesized and evaluated:

fadDD35 from M. tuberculosis HR7Rv [NP_217021].

yhfL from B. subtilis [NP_388908].

fadD1 from P. aeruginosa PAO1 [NP_251989].

fadD homolog, encoding Faa3p from Saccharomyces cerevisiae [NP_012257].

Additional fatty acid acyl-CoA synthases from eukaryotic organisms,which can be used to produce acyl-CoA as well as fatty acid derivatives,include those described in Shockey et al., Plant Physiol., 129:1710-1722, 2002 (Arabidopsis), Caviglia et al., J. Biol. Chem., 279:1163-1169, 2004 (rat), and Knoll et al., J. Biol. Chem.,269(23):16348-56, 1994 (yeast). Gene sequences encoding thesesynthetases are known in the art. See, e.g., Johnson et al., J. Biol.Chem., 269: 18037-18046, 1994; Shockey et al., Plant Physiol., 129:1710-1722, 2002; Black et al., J. Biol. Chem., 267: 25513-25520, 1992.These eukaryotic acyl-CoA synthases, despite lacking in high homology toE. coli FadD sequences, can complement FadD activity in E. coli FadDknockouts.

A. Acyl-CoA to Fatty Alcohol

1. Conversion of Acyl-CoA to Fatty Alcohol

Acyl-CoA is reduced to a fatty aldehyde by an NADH-dependent acyl-CoAreductase (e.g., Acr1). The fatty aldehyde is then reduced to a fattyalcohol by an NADPH-dependent alcohol dehydrogenase (e.g., YqhD).Alternatively, fatty alcohol forming acyl-CoA reductase (FAR) catalyzesthe reduction of an acyl-CoA into a fatty alcohol and CoASH. FAR usesNADH or NADPH as a cofactor in this four-electron reduction. Althoughthe alcohol-generating FAR reactions proceed through an aldehydeintermediate, a free aldehyde is not released. Thus, the alcohol-formingFARs are distinct from the enzymes that carry out two-electronreductions of acyl-CoA and yield free fatty aldehyde as a product. (SeeCheng and Russell, J. Biol. Chem., 279(36):37789-37797, 2004; Metz etal., Plant Physiol., 122:635-644, 2000).

2. Modifying the Fatty Acid Biosynthetic Pathways to Increase Conversionof Acyl-CoA to Fatty Alcohol

Production hosts can be engineered using known polypeptides to producefatty alcohols from acyl-CoA. One method of making fatty alcoholsinvolves increasing the expression of, or expressing more active formsof, fatty alcohol forming acyl-CoA reductases (encoded by a gene such asacr1, EC 1.2.1.50/1.1.1), acyl-CoA reductases (EC 1.2.1.50), and/oralcohol dehydrogenases (EC 1.1.1.1).

Fatty alcohols are often described as hydrocarbon-based surfactants.They also serve as suitable components of surfactants. For surfactantproduction, the production host is modified so that it produces asurfactant from a renewable carbon source. Such a production hostincludes a first exogenous polynucleotide sequence encoding a proteincapable of converting a fatty acid to a fatty aldehyde and a secondexogenous polynucleotide sequence encoding a protein capable ofconverting a fatty aldehyde to an alcohol. In some examples, the firstexogenous polynucleotide sequence encodes a fatty acid reductase. In oneembodiment, the second exogenous polynucleotide sequence encodesmammalian microsomal aldehyde reductase or long-chain aldehydedehydrogenase. In a further example, the first and second exogenouspolynucleotide sequences are from Arthrobacter AK 19, Rhodotorulaglutinins, Acinetobacter sp. strain M-1, or Candida lipolytica. In oneembodiment, the first and second heterologous polynucleotide sequencesform a multienzyme complex from Acinetobacter sp. strain M-1 or fromCandida lipolytica.

Additional sources of heterologous DNA sequences encoding fatty acid tolong chain alcohol converting proteins that can be used in surfactantproduction include, but are not limited to, Mortierella alpina (ATCC32222), Cryptococcus curvatus, (also referred to as Apiotricumcurvatum), Alcanivorax jadensis (T9T=DSM 12718=ATCC 700854),Acinetobacter sp. HO1-N (ATCC 14987) and Rhodococcus opacus (PD630 DSMZ44193).

In one example, the fatty acid derivative is a saturated or unsaturatedsurfactant product having a carbon chain length of about 6 to about 36carbon atoms, about 8 to about 30 carbon atoms, about 10 to about 26carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 16carbon atoms. In another example, the surfactant product has a carbonchain length of about 10 to about 18 carbon atoms, or about 12 to about14 carbon atoms.

Suitable production hosts for producing surfactants include eukaryoticor prokaryotic microorganisms. Exemplary production hosts includeArthrobacter AK 19, Rhodotorula glutinins, Acinetobacter sp. strain M-1,Arabidopsis thalania, Candida lipolytica, Saccharomyces cerevisiae,cyanobacteria such as Synechocystis spp. and Synechococcus spp., Algaesuch as Clamydomonas, and E. coli engineered to overexpress acetyl-CoAcarboxylase. Production hosts that demonstrate an innate ability tosynthesize high levels of surfactant precursors in the form of lipidsand oils, such as Rhodococcus opacus, Arthrobacter AK 19, Rhodotorulaglutinins, E. coli engineered to express acetyl CoA carboxylase, andother oleaginous cyanobacteria, bacteria, yeast, and fungi can also beused.

B. Fatty Alcohols to Fatty Esters

Production hosts can be engineered using known polypeptides to producefatty esters of various lengths. One method of making fatty estersincludes increasing the expression of, or expressing more active formsof, one or more alcohol O-acetyltransferase peptides (EC 2.3.1.84).These peptides catalyze the acetylation of an alcohol by converting anacetyl-CoA and an alcohol to a CoA and an ester. In some examples, thealcohol O-acetyltransferase peptides can be expressed in conjunctionwith selected thioesterase peptides, FAS peptides, and fatty alcoholforming peptides, thus allowing the control of carbon chain lengths,saturation levels, and degrees of branching. In some cases, the bkdoperon can be coexpressed in order to produce branched fatty acidprecursors.

As used herein, alcohol O-acetyltransferase peptides include peptides inenzyme classification number EC 2.3.1.84, as well as any other peptidescapable of catalyzing the conversion of an acetyl-CoA and an alcohol toform a CoA and an ester. Additionally, one of ordinary skill in the artwill appreciate that alcohol O-acetyltransferase peptides can alsocatalyze other reactions.

For example, some alcohol O-acetyltransferase peptides can accept othersubstrates in addition to fatty alcohols and/or acetyl-CoA thioesters,such as other alcohols and other acyl-CoA thioesters. Such non-specificor divergent-specificity alcohol O-acetyltransferase peptides are,therefore, also included. Various alcohol O-acetyltransferase peptidesequences are publicly available. Assays for measuring the activity ofalcohol O-acetyltransferase peptides are known in the art. Moreover,O-acyltransferases can be engineered to impart new activities and/orspecificities for the donor acyl group or acceptor alcohol moiety.Engineered enzymes can be generated through well documented rational andevolutionary approaches.

C. Acyl-CoA to Fatty Esters

1. Production of Fatty Esters

Fatty esters are synthesized by an acyl-CoA:fatty alcoholacyltransferase (e.g., ester synthase), which conjugates a long chainfatty alcohol to a fatty acyl-CoA via an ester linkage. Ester synthasesand the encoding genes are known from the jojoba plant and the bacteriumAcinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticusADP1). The bacterial ester synthase is a bifunctional enzyme, exhibitingester synthase activity and the ability to form triacylglycerols fromdiacylglycerol substrates and fatty acyl-COAs (acyl-CoA:diglycerolacyltransferase (DGAT) activity). The gene wax/dgat encodes both estersynthase and DGAT. See Cheng et al., J. Biol. Chem.,279(36):37798-37807, 2004; Kalscheuer and Steinbuchel, J. Biol. Chem.,278:8075-8082, 2003. Ester synthases can also be used to produce certainfatty esters that can be used as a fuel, such as biodiesel, as describedherein.

2. Modifying the Fatty Acid Biosynthetic Pathway to Produce Fatty EstersUsing Ester Synthase

The production of fatty esters, including waxes, from acyl-CoA andalcohols, can be engineered using known polypeptides. One method ofmaking fatty esters includes increasing the expression of, or expressingmore active forms of, one or more ester synthases (EC 2.3.1.20,2.3.1.75). Various ester synthase peptide sequences are publiclyavailable. Methods of determining ester synthase activity are providedin U.S. Pat. No. 7,118,896, which is herein incorporated by reference inits entirety.

In certain embodiments, if the desired product is an ester-basedbiofuel, a production host can be modified such that it produces anester from a renewable energy source. Such a production host includes anexogenous genes encoding an ester synthase that is expressed so as toconfer upon said production host the ability to synthesize a saturated,unsaturated, or branched fatty ester from a renewable energy source. Insome embodiments, the organism can also express genes encoding thefollowing exemplary proteins: fatty acid elongases, acyl-CoA reductases,acyltransferases, ester synthases, fatty acyl transferases,diacylglycerol acyltransferases, thioesterases, and/or acyl-coA waxalcohol acyltransferases. In an alternate embodiment, the organismexpresses a gene encoding a bifunctional estersynthase/acyl-CoA:diacylglycerol acyltransferase. For example, thebifunctional ester synthase/acyl-CoA:diacylglycerol acyltransferase canbe selected from the multi-enzyme complexes from Simmondsia chinensis,Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticusADP1), Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacterjadensis, Arabidopsis thaliana, or Alcaligenes eutrophus (later renamedRalstonia eutropha). In one embodiment, the fatty acid elongases,acyl-CoA reductases, or wax synthases are obtained and/or derived from amulti-enzyme complex from Alcaligenes eutrophus (later renamed Ralstoniaeutropha) or other organisms known in the literature to produce esterssuch as wax or fatty esters.

Additional sources of heterologous DNA sequences encoding estersynthesis proteins useful in fatty ester production include, but are notlimited to, Mortierella alpina (e.g., ATCC 32222), Cryptococcus curvatus(also referred to as Apiotricum curvatum), Alcanivorax jadensis (e.g.,T9T=DSM 12718=ATCC 700854), Acinetobacter sp. HO1-N, (e.g., ATCC 14987)and Rhodococcus opacus (e.g., PD630, DSMZ 44193).

Useful production hosts for producing fatty esters can be eukaryotic orprokaryotic microorganisms. Non-limiting examples of production hostsfor producing fatty esters include Saccharomyces cerevisiae,Synechococcus, Synechocystis, Clamydomonas, Candida lipolytica, E. coli,Arthrobacter AK 19, Rhodotorula glutinins, Acinetobacter sp. strain M-1,Candida lipolytica, and other oleaginous microorganisms.

In one example, the ester synthase from Acinetobacter sp. ADP1 at locusAA017391 (described in Kalscheuer and Steinbuchel, J. Biol. Chem.,278:8075-8082, 2003, herein incorporated by reference) is used. Inanother example, the ester synthase from Simmondsia chinensis at locusAAD38041 is used.

In certain embodiments, the esters produced in accordance with themethods and compositions herein are secreted or released from the hostcells, and thus can be recovered extracellularly. Optionally, an esterexporter such as a member of the FATP family can be used to facilitatethe release of esters into the extracellular environment. A non-limitingexample of a suitable ester exporter is fatty acid (long chain)transport protein CG7400-PA, isoform A, from Drosophila melanogaster, atlocus NP_524723.

D. Acyl-ACP, Acyl-CoA to Hydrocarbon

1. Hydrocarbons from Particular Microorganisms

A diverse set of microorganisms are known to produce hydrocarbons, suchas alkanes, olefins, and isoprenoids. Many of these hydrocarbons arederived from fatty acid biosynthesis. The production of thesehydrocarbons can be controlled by controlling the genes associated withfatty acid biosynthesis in the native production hosts.

For example, hydrocarbon biosynthesis in the algae Botryococcus brauniioccurs via the decarbonylation of fatty aldehydes. The fatty aldehydesare produced by the reduction of fatty acyl thioesters by an enzyme suchas a fatty acyl-CoA reductase. Thus, the structure of the final alkanescan be controlled by engineering B. braunii to express specific genes,such as thioesterases, which control the chain length of the fatty acidsbeing channeled into alkane biosynthesis. Expressing the enzymes thatresult in branched chain fatty acid biosynthesis in B. braunii willresult in the production of branched chain alkanes. Introduction ofgenes affecting the production of desaturated fatty acids will result inthe production of olefins. Further combinations of these genes canprovide further control over the final structure of the hydrocarbonsthat will be produced.

To produce higher levels of native or engineered hydrocarbons, the genesinvolved in the biosynthesis of fatty acids and their precursors, or thedegradation of other products can be expressed, overexpressed, orattenuated. Each of these approaches can be applied to the production ofalkanes in Vibrio furnissii M1 and other Vibrio furnissii strains, whichproduce alkanes through the reduction of fatty alcohols. In addition toVibrio furnissii, other alkane producing organisms that utilize thefatty acid pathway can be used.

Each of these approaches can also be applied to the production of theolefins produced by strains of Micrococcus leuteus, Stenotrophomonasmaltophilia, and related microorganisms. These microorganisms producelong chain olefins that are derived from the head-to-head condensationof fatty acid precursors. Controlling the structure and level of thefatty acid precursors using the methods described herein will result inthe formation of olefins of different chain lengths, branchingcharacteristics, and levels of saturation.

Cyanobacteria can also be used as suitable production hosts for theproduction of fatty acid derivatives such as fatty alcohols, fattyesters, and hydrocarbons. For example, Synechocystis sp. PCC6803 andSynechococcus elongatus PCC7942 can serve as production hosts and can beengineered using standard molecular biology techniques (Thiel, Geneticanalysis of cyanobacteria, in THE MOLECULAR BIOLOGY OF CYANOBACTERIA,ADVANCES IN PHOTOSYNTHESIS AND RESPIRATION 581-611 (Kluwer AcademicPublishers), 1994; Koksharova and Wolk, Appl. Microbiol. Biotechnol.,58: 123-137, 2002, the contents of which are incorporated by referenceherein. Fatty acid biosynthesis genes can be readily identified andisolated in these organisms.

Furthermore, many cyanobacteria are natural producers of hydrocarbons,such as heptadecane, and therefore contain hydrocarbon biosynthesisgenes that can be deregulated and overexpressed in conjunction withmanipulating their fatty acid biosynthesis genes, in order to increasehydrocarbon production.

Unlike other bacteria, some cyanobacteria (e.g., Synechocystis sp.PCC6803) contain polyunsaturated fatty acids in their lipids (Murata,Plant cell Physiol., 33: 933-941, 1992), and thus have the inherentcapability to produce polyunsaturated fatty acid derivatives. Mostimportantly, cyanobacteria are photosynthetic organisms that synthesizeall cellular carbon by harvesting sun light and fixing carbon dioxide.Therefore, fatty acid derivatives produced in cyanobacteria are directlyderived from CO₂.

2. Producing Hydrocarbons from Reduction of Primary Alcohols

Hydrocarbons can also be produced using evolved oxidoreductases for thereduction of primary alcohols. Using primary fatty alcohols to producealkanes in microorganisms, such as Vibrio fumissii M1, is known. See,e.g., Park, J. Bacteriol., 187:1426-1429, 2005, the content of which isincorporated herein by reference. One example of an oxidoreductase thatcan be used to produce hydrocarbons from fatty alcohols isNAD(P)H-dependent oxidoreductase. Synthetic NAD(P)H dependentoxidoreductases can be produced through the use of evolutionaryengineering and can be expressed in production hosts to produce fattyacid derivatives.

The process of “evolving” a fatty alcohol reductase to have the desiredactivity is known and practiced by those skilled in the art (Kolkman andStemmer, Nat. Biotechnol., 19:423-8, 2001; Ness et al., Adv. ProteinChem., 55:261-92, 2000; Minshull and Stemmer, Curr. Opin. Chem. Biol.,3:284-90, 1999; Huisman and Gray, Curr. Opin. Biotechnol., 13:352-8,2002; U.S. Patent Publication No. 2006/0195947), the contents of all ofwhich are incorporated herein by reference.

A library of NAD(P)H-dependent oxidoreductases is generated by standardmethods, such as error-prone PCR, site-specific random mutagenesis,site-specific saturation mutagenesis, or site-directed specificmutagenesis. Additionally, a library can be created through the“shuffling” of naturally-occurring NAD(P)H-dependent oxidoreductaseencoding sequences. The library is expressed in a suitable productionhost, such as an E. coli. Individual colonies expressing a differentmember of the oxidoreductase library are then analyzed for expression ofan oxidoreductase that can catalyze the reduction of a fatty alcohol.

For example, each cell can be assayed as a whole cell bioconversion, acell extract, or a permeabilized cell. Enzymes purified from the cellcan be analyzed as well. Fatty alcohol reductases are identified byspectrophotometrically or fluorometrically monitoring the fattyalcohol-dependent oxidation of NAD(P)H. Production of alkanes ismonitored by GC-MS, TLC, or other suitable methods.

An oxidoreductase identified in this manner is used to produce alkanes,alkenes, and related branched hydrocarbons. This is achieved either invitro or in vivo. The latter is achieved by expressing the evolved fattyalcohol reductase gene in an organism that produces fatty alcohols, suchas the ones described herein. The fatty alcohols act as substrates forthe alcohol reductase, which produces alkanes Other oxidoreductases canalso be engineered to catalyze this reaction, such as those that usemolecular hydrogen, glutathione, FADH, or other reductive coenzymes.

3. Conversion of Acyl-ACP to Ketone and/or Olefins

Acyl-ACP can be converted to a ketone and/or an internal olefin by theaction of acyl condensing enzymes, as described in PCT Publication No.2008/147781 A2, the disclosures of which are incorporated herein byreference. As described in the '781 publication, acyl-condensingpeptides include peptides that are capable of catalyzing thecondensation of acyl-ACP, acyl-CoA, acyl-AMP, fatty acids, and mixturesthereof using the methods described therein. In some embodiments, theseacyl-condensing peptides have high, medium, or low substratespecificity. In certain examples, the acyl-condensing peptides are moresubstrate specific and will only accept substrates of a specific chainlength. Additionally, one of ordinary skill in the art will appreciatethat some acyl-condensing peptides will catalyze other reactions aswell. Examples of acyl-condensing enzymes are disclosed in the '781publication. In addition, the '781 publication describes adenylatingproteins, dehydratases, and dehydrogenases that can be used in theproduction of hydrocarbons such as internal olefins.

Recombinant organisms can be engineered using polynucleotides andproteins, for example, those disclosed in the '781 publication, toproduce hydrocarbons and aliphatic ketones that have defined structuralcharacteristics (e.g., degrees of branching, levels of saturation, orcarbon chain lengths). One method of making hydrocarbons involvesincreasing the expression of, or expressing more active forms of, one ormore acyl-condensing enzymes (enzymes that condense two or more ofacyl-CoA, acyl-ACP, acyl-AMP, acyl-ester, fatty acid, or mixturesthereof). One of ordinary skill in the art will appreciate that theproducts produced from such condensation reactions vary depending on theacyl chain that is condensed. Products that can be produced include, forexample, hydrocarbons and hydrocarbon intermediates, such as aliphaticketones. The aliphatic ketones, hydrocarbons, and hydrocarbonintermediates can be engineered to have specific carbon chaincharacteristics by expressing various enzymes or attenuating theexpression of various enzymes in the recombinant organism. According tothe present invention, the mutant thioesterases of the invention can beused to manipulate the range of acyl species carbon chain lengths. Thus,by using a mutant thioesterase having a particular substrate specificityor selectivity, it is possible to affect the downstream reactions so asto result in a predetermined olefin or ketone product profile.

4. Conversion of Fatty Acid to Aldehyde

Fatty acids resulting from thioesterase cleavage can be converted to analdehyde by the action of the carboxylic acid reductase gene. Aldehydescan be useful products in themselves, or they can serve as substratesfor further enzymatic catalysis reactions, for example, in theproduction of fatty alcohols via an enzymatic reaction of alcoholdehydrogenase, or in the production of alkanes via an enzymatic reactionof decarbonylases. According to the compositions and methods herein, thefatty acid substrates of the carboxylic acid reductase can bemanipulated so as to achieve a predetermined product profile in thealdehyde or fatty alcohol product.

E. Release of Fatty Acid Derivatives—with or without Transport Proteins

As described herein, the fatty acid derivatives produced in accordancewith the methods, compositions, vectors, and host cells herein can besecreted or spontaneously released so as to allow the recovery of thefatty acid derivative products extracellularly. The speed of spontaneoussecretion may or may not be sufficiently fast, and the level of releasemay or may not be sufficiently complete. Therefore, optionally,transport proteins can be used to facilitate export of fatty acidderivatives out of the production host. Transport and efflux proteinsare known to excrete a large variety of compounds, and can naturally bemodified to be selective for particular types of fatty acid derivatives.Non-limiting examples of suitable transport proteins are ATP-BindingCassette (ABC) transport proteins, efflux proteins, and fatty acidtransporter proteins (FATP). Additional non-limiting examples ofsuitable transport proteins include the ABC transport proteins fromorganisms such as Caenorhabditis elegans, Arabidopsis thalania,Alkaligenes eutrophus, and Rhodococcus erythropolis. Exemplary ABCtransport proteins include CER5, AtMRP5, AmiS2, or AtPGP1. In apreferred embodiment, the ABC transport protein is a CER5 (e.g.,AY734542)). Vectors containing genes that express suitable transportproteins can be inserted into protein production hosts to increase ordrive the release of fatty acid derivatives.

Production of fatty acid derivative products according to the presentinvention does not require transport or efflux protein modification andit is possible to select production hosts for their endogenous abilityto release fatty acid derivatives. Furthermore, simply by constructinghost cells according to the present disclosure, for example, fatty acidderivative products that are otherwise not known to be secreted can besecreted or spontaneously released. The efficiency of product productionand release into the fermentation broth can be expressed as a ratio ofintracellular product to extracellular product. In some examples, theratio can be about 100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1,1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40 or 1:50.

II. Selection of Carbon Chain Characteristics of Fatty Acid Derivatives

Fatty acid derivatives with particular branch points, levels ofsaturation, carbon chain lengths, and ester characteristics can beproduced as desired. Microorganisms that naturally produce particularderivatives can be selected as production hosts, and in certaincircumstances, endogenous enzymes therein can be manipulated to producefatty acid derivatives of desirable characteristics. Alternatively,genes that express enzymes that will produce particular fatty acidderivatives can be suitably inserted into the production hostmicroorganisms.

In some examples, expression of exogenous FAS genes originating fromdifferent species or engineered variants can be achieved in a productionhost, resulting in the biosynthesis of fatty acids that are structurallydifferent (in, for example, lengths, levels of branching, degrees ofunsaturation, etc.) from those of the native production host. Theseheterologous gene products can also be selected or engineered to beunaffected by the natural regulatory mechanisms in the production hostcells, and as such allowing control of the production of the desiredcommercial product. For example, the FAS enzymes from Bacillus subtilis,Saccharomyces cerevisiae, Streptomyces spp., Ralstonia, Rhodococcus,Corynebacteria, Brevibacteria, Mycobacteria, oleaginous yeast, or thelike can be expressed in a suitable production host. The expression ofsuch exogenous enzymes will alter the structure of the fatty acidproduced.

When a production host is engineered to produce a fatty acid with aspecific level of unsaturation, branching, or carbon chain length, theresulting engineered fatty acid can be used in the production of fattyacid derivatives. Fatty acid derivatives generated from such productionhosts can display the characteristics of the engineered fatty acid.

For example, a production host can be engineered to make branched, shortchain fatty acids, which can then be used by the production host toproduce branched, short chain fatty alcohols. Similarly, a hydrocarboncan be produced by engineering a production host to produce a fatty acidhaving a defined level of branching, unsaturation, and/or carbon chainlength, and thus producing a homogeneous hydrocarbon population.Additional steps can be employed to improve the homogeneity of theresulting product. For example, when an unsaturated alcohol, fattyester, or hydrocarbon is desired, the production host organism can beengineered to produce low levels of saturated fatty acids, and inaddition can be modified to express an additional desaturase and thuslessen the production of saturated product.

A. Branched and Cyclic Moieties

1. Engineering Branched and Cyclic Fatty Acid Derivatives

Fatty acids are key intermediates in the production of fatty acidderivatives. Fatty acid derivatives containing branch points, cyclicmoieties, and combinations thereof can be prepared using branched orcyclic fatty acids.

For example, E. coli naturally produces straight chain fatty acids(sFAs). To engineer E. coli to produce branched chain fatty acids(brFAs), several genes that provide branched precursors (e.g., a bkdoperon) can be introduced into the production host and expressed toallow initiation of fatty acid biosynthesis from branched precursors(e.g., fabH). The bkd, ilv, icm, and fab gene families can be expressedor overexpressed to produce branched chain fatty acid derivatives.Similarly, to produce cyclic fatty acids, genes that provide cyclicprecursors can be introduced into the production host and expressed toallow initiation of fatty acid biosynthesis from cyclic precursors. Theans, chc, and plm gene families can be expressed or overexpressed toproduce cyclic fatty acids.

Additionally, a production host can be engineered to express genesencoding proteins for the elongation of brFAs (e.g., genes encoding ACP,FabF, etc.) and/or to delete or attenuate the corresponding E. coligenes that normally lead to sFAs. In this regard, endogenous genes thatwould compete with the introduced genes (e.g., fabH, fabF) are deletedor attenuated.

The branched acyl-CoA (e.g., 2-methyl-butyryl-CoA, isovaleryl-CoA,isobutyryl-CoA, etc.) are the precursors of brFA. In most microorganismscontaining brFA, the brFA are synthesized in two steps from branchedamino acids (e.g., isoleucine, leucine, or valine) (Kadena, Microbiol.Rev., 55:288, 1991). A production host can be engineered to express oroverexpress one or more of the enzymes involved in these two steps toproduce brFAs, or to over-produce brFAs. For example, the productionhost may have an endogenous enzyme that can accomplish one step leadingto brFA, therefore only genes encoding enzymes involved in the secondstep need to be introduced recombinantly.

The mutant thioesterases of the invention can be engineered to have oneor more altered properties, for example, altered specificity and/orincreased activity (e.g., catalytic rate), with regard to branched orcyclic chain acyl-CoA or acyl-ACP compounds described herein.Accordingly the recombinant cell producing fatty acid derivatives can bemade to preferentially produce a desired branched or cyclic chain fattyacid derivative product that may have high value as an end product.

2. Formation of Branched Fatty Acids and Branched Fatty Acid Derivatives

The first step in forming brFAs is the production of the correspondinga-keto acids by a branched-chain amino acid aminotransferase. Productionhosts can endogenously include genes encoding such enzymes, oralternatively, such genes can be recombinantly introduced. E. coli, forexample, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42;GenBank Accession No. YP_026247). In some production hosts, aheterologous branched-chain amino acid aminotransferase may not beexpressed. However, E. coli IlvE or any other branched-chain amino acidaminotransferase (e.g., IlvE from Lactococcus lactic (GenBank AccessionNo. AAF34406), IlvE from Pseudomonas putida (GenBank Accession No.NP_745648), or IlvE from Streptomyces coelicolor (GenBank Accession No.NP_629657)), if not endogenous, can be introduced. If theaminotransferase reaction is rate limiting in brFA biosynthesis in thechosen production host organism, then the aminotransferase can beoverexpressed.

The second step is the oxidative decarboxylation of the a-keto acids tothe corresponding branched-chain acyl-CoA. This reaction can becatalyzed by a branched-chain a-keto acid dehydrogenase complex (bkd; EC1.2.4.4.) (Denoya et al., J. Bacteriol., 177:3504, 1995), which consistsof E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase) and E3(dihydrolipoyl dehydrogenase) subunits. These branched-chain a-keto aciddehydrogenase complexes are similar to pyruvate and a-ketoglutaratedehydrogenase complexes. Every microorganism that possesses brFAs and/orgrows on branched-chain amino acids can be used as a source to isolatebkd genes for expression in production hosts such as, for example, E.coli. Furthermore, E. coli has the E3 component as part of its pyruvatedehydrogenase complex (encoded by, for example, lpd, EC 1.8.1.4, GenBankAccession No. NP_414658), thus it can be sufficient to only express theE1α/β and E2 bkd genes. Table 2 recites non-limiting examples of bkdgenes from several microorganisms that can be recombinantly introducedand expressed in a production host to provide branched-chain acyl-CoAprecursors. Microorganisms having such bkd genes can also be used asproduction hosts.

TABLE 2 Bkd genes from selected microorganisms GenBank Organism GeneAccession No. Streptomyces coelicolor bkdA1 (E1α) NP_628006 bkdB1 (E1β)NP_628005 bkdC1 (E2) NP_638004 Streptomyces coelicolor bkdA2 (E1α)NP_733618 bkdB2 (E1β) NP_628019 bkdC2 (E2) NP_628018 Streptomycesavermitilis bkdA (E1a) BAC72074 bkdB (E1b) BAC72075 bkdC (E2) BAC72076Streptomyces avermitilis bkdF (E1α) BAC72088 bkdG (E1β) BAC72089 bkdH(E2) BAC72090 Bacillus subtilis bkdAA (E1α) NP_390288 bkdAB (E1β)NP_390288 bkdB (E2) NP_390288 Pseudomonas putida bkdA1 (E1α) AAA65614bkdA2 (E1β) AAA65615 bkdC (E2) AAA65617

In another example, isobutyryl-CoA can be made in a production host, forexample in E. coli, through the coexpression of a crotonyl-CoA reductase(Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunitIcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds,J. Bacteriol., 179:5157, 1997). Crotonyl-CoA is an intermediate in fattyacid biosynthesis in E. coli and other microorganisms. Non-limitingexamples of ccr and icm genes from selected microorganisms are given inTable 3.

TABLE 3 ccr and icm genes from selected microorganisms GenBank OrganismGene Accession No. Streptomyces coelicolor ccr NP_630556 icmA NP_629554icmE NP_630904 Streptomyces cinnamonensis ccr AAD53915 icmA AAC08713icmB AJ246005

In addition to expression of the bkd genes, the initiation of brFAbiosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase III(FabH, EC 2.3.1.41) with specificity for branched chain acyl-COAs (Li etal., J. Bacteriol., 187:3795-3799, 2005). Non-limiting examples of suchFabH enzymes are listed in Table 4. fabH genes that are involved infatty acid biosynthesis of any brFA-containing microorganism can beexpressed in a production host. The Bkd and FabH enzymes from productionhosts that do not naturally make brFA may not support brFA production,therefore Bkd and FabH can be expressed recombinantly. Vectorscontaining the bkd and fabH genes can be inserted into such a productionhost. Similarly, the endogenous level of Bkd and FabH production may notbe sufficient to produce brFA, therefore, they can be over-expressed.Additionally, other components of fatty acid biosynthesis pathway can beexpressed or over-expressed, such as acyl carrier proteins (ACPs) andβ-ketoacyl-acyl-carrier-protein synthase II (encoded by fabF, EC2.3.1.41) (non-limiting examples of candidates are listed in Table 4).In addition to expressing these genes, some genes in the endogenousfatty acid biosynthesis pathway may be attenuated in the productionhost. Genes encoding enzymes that compete for substrate(s) with theenzymes of the pathway that result in brFA production can be attenuatedto increase brFA production. For example, in E. coli the most likelycandidates to interfere with brFA biosynthesis are fabH (GenBankAccession No. NP_415609) and/or fabF genes (GenBank Accession No.NP_415613).

TABLE 4 fabH, ACP and fabF genes from selected microorganisms with brFAsGenBank Organism Gene Accession No. Streptomyces coelicolor fabH1NP_626634 ACP NP_626635 fabF NP_626636 Streptomyces avermitilis fabH3NP_823466 fabC3 (ACP) NP_823467 fabF NP_823468 Bacillus subtilis fabH_ANP_389015 fabH_B NP_388898 ACP NP_389474 fabF NP_389016 Stenotrophomonasmaltophilia SmalDRAFT_0818 ZP_01643059 (fabH) SmalDRAFT_0821 ZP_01643063(ACP) SmalDRAFT_0822 ZP_01643064 (fabF) Legionella pneumophila FabHYP_123672 ACP YP_123675 fabF YP_123676

As mentioned above, branched chain alcohols can be produced through thecombination of expressing genes that support brFA synthesis and alcoholsynthesis. For example, when a gene encoding an alcohol reductase, suchas acrl from Acinetobacter baylyi ADP1, is coexpressed with a bkd operonin an E. coli host cell, the host cell can synthesize isopentanol,isobutanol, or 2-methyl butanol. Similarly, when acrl is coexpressedwith ccrlicm genes in an E. coli host cell, the host cell can synthesizeisobutanol.

3. Formation of Cyclic Fatty Acids and Cyclic Fatty Acid Derivatives

To convert a production host such as an E. coli into an organism capableof synthesizing ω-cyclic fatty acids (cyFA), a gene that provides thecyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA) (Cropp et al., NatureBiotech., 18:980-983, 2000) is introduced and expressed in theproduction host. A similar conversion is possible for other productionhosts, for example, bacteria, yeast and filamentous fungi.

Non-limiting examples of genes that provide CHC-CoA in E. coli include:ansJ, ansK, ansL, chcA, and ansM from the ansatrienin gene cluster ofStreptomyces collinus (Chen et al., Eur. J. Biochem., 261: 98-107,1999), or plmJ, plmK, plmL, chcA, and plmM from the phoslactomycin Bgene cluster of Streptomyces sp. HK803 (Palaniappan et al., J. Biol.Chem., 278:35552-35557, 2003) together with the chcB gene (Patton etal., Biochem., 39:7595-7604, 2000) from S. collinus, S. avermitilis, orS. coelicolor (see Table 5 for GenBank Accession numbers). The geneslisted above in Table 4 can then be expressed to allow initiation andelongation of w-cyclic fatty acids. Alternatively, the homologous genescan be isolated from microorganisms that make cyFA and expressed in E.coli.

TABLE 5 Genes for the synthesis of CHC-CoA GenBank Organism GeneAccession No. Streptomyces collinus ansJK U72144* ansL chcA ansM chcBAF268489 Streptomyces sp. HK803 pmlJK AAQ84158 pmlL AAQ84159 chcAAAQ84160 pmlM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292Streptomyces avermitilis chcB/caiD NP_629292 *Only chcA is annotated inGenBank entry U72144, ansJKLM are according to Chen et al., Eur. J.Biochem., 261: 98-107, 1999.

The genes listed in Table 4 (fabH, ACP, and fabF) are sufficient toallow initiation and elongation of ω-cyclic fatty acids because theytypically have broad substrate specificity. If the coexpression of anyof these genes with the ansJKLM/chcAB or pmlJKLM/chcAB genes from Table5 does not yield cyFA, then fabH, ACP, and/or fabF homologs frommicroorganisms that make cyFAs can be isolated (e.g., by usingdegenerate PCR primers or heterologous DNA sequence probes) andcoexpressed. Table 6 lists non-limiting examples of microorganisms thatcontain ω-cyclic fatty acids.

TABLE 6 Non-limiting examples of microorganisms that contain ω-cyclicfatty acids Organism Reference Curtobacterium pusillum ATCC19096Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillusacidocaldarius ATCC27009 Alicyclobacillus cycloheptanicus * Moore, J.Org. Chem., 62: pp. 2173, 1997. * Uses cycloheptylcarbonyl-CoA and notcyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.

B. Saturation

Fatty acids are key intermediates in the production of fatty acidderivatives. The degrees of saturation in fatty acid derivatives can becontrolled by regulating the degrees of saturation of the fatty acidintermediates. The sfa, gns, and fab families of genes can be expressedor overexpressed to control the saturation of fatty acids.

Production hosts can be engineered to produce unsaturated fatty acids byengineering the production host to overexpress fabB, or by growing theproduction host at low temperatures (e.g., less than 37° C.). FabB haspreference for cis-δ³decenoyl-ACP, and results in unsaturated fatty acidproduction in E. coli. Overexpression of the fabB gene results in theproduction of a significant percentage of unsaturated fatty acids (deMendoza et al., J. Biol. Chem., 258:2098-101, 1983). The fabB gene canbe inserted into and expressed in production hosts not naturally havingthe gene. These unsaturated fatty acids can then be used asintermediates in the production hosts that are engineered to producefatty acid derivatives, such as fatty alcohols, fatty esters, waxes,olefins, alkanes, and the like.

Alternatively, repressors of fatty acid biosynthesis, for example, arepressor (GenBank Accession No. NP_418398) encoded by fabB, can bedeleted. This will also result in increased unsaturated fatty acidproduction in E. coli (Zhang et al., J. Biol. Chem., 277:15558, 2002).Similar deletions can be made in other production hosts. Furtherincrease in unsaturated fatty acids may be achieved, for example, byoverexpression of fabM (encoding trans-2, cis-3-decenoyl-ACP isomerase,GenBank Accession No. DAA05501) and controlled expression of fabK(encoding trans-2-enoyl-ACP reductase II, GenBank Accession No.NP_357969) from Streptococcus pneumoniae (Marrakchi et al., J. Biol.Chem., 277: 44809, 2002), while deleting E. coli fabs (encodingtrans-2-enoyl-ACP reductase, GenBank Accession No. NP_415804).Additionally, to increase the percentage of unsaturated fatty esters,the production host can also overexpress fabB (encoding β-ketoacyl-ACPsynthase I, GenBank Accession No. BAA16180, EC:2.3.1.41), sfa (encodinga suppressor of fabA, GenBank Accession No. AAC44390), and gnsA and gnsB(both encoding secG null mutant suppressors, GenBank Accession No.ABD18647.1 and GenBank Accession No. AAC74076.1, respectively). In someexamples, the endogenous fabF gene can be attenuated, thus increasingthe percentage of palmitoleate (C_(16:1)) produced.

The mutant thioesterases of the invention can be engineered to havealtered properties, for example, altered specificity and/or increasedactivity, with regard to substituted or unsubstituted acyl-CoA oracyl-ACP compounds that are prepared as described herein. Accordinglythe recombinant cell producing the fatty acid derivatives can be made topreferentially produce a desired saturation profile in a fatty acidderivative product that may have high value as an end product.

C. Chain Lengths and Ester Characteristics

1. Chain Lengths and Production of Odd-Numbered Chains

The methods described herein permit production of fatty esters and fattyacid derivatives of varied chain lengths by selecting a suitable mutantthioesterase that has specificity and/or selectivity for a substrate ofa specific carbon chain length. By expressing the specificthioesterases, fatty acids and fatty acid derivatives having desiredcarbon chain lengths can be produced. In some embodiments, an endogenousthioesterase can be mutated using known genomic alteration techniques.Or, a gene encoding a particular thioesterase can be heterologouslyintroduced into a production host such that a fatty acid or fatty acidderivative of a particular carbon chain length is produced. In certainembodiments, expression of endogenous thioesterases is suppressed. Themutant thioesterases of the invention can be engineered to have alteredproperties, for example, altered specificity and/or increased activity,with regard to specific chain lengths of acyl-CoA or acyl-ACP compoundsdescribed herein. Accordingly, the recombinant cell producing the fattyacid derivatives can be made to preferentially produce a fatty acidderivative product with the desired chain length and/or high value as anend product.

In one embodiment, the fatty acid derivative contains a carbon chain ofabout 4 to 36 carbon atoms, about 6 to 32 carbon atoms, about 10 to 30carbon atoms, about 10 to 18 carbon atoms, about 24 to 32 carbon atoms,about 26 to 30 carbon atoms, about 26 to 32 carbon atoms, about 5 to 10carbon atoms, about 10 to 16 carbon atoms, or about 12 to 18 carbonatoms. In an alternate embodiment, the fatty acid derivative contains acarbon chain less than about 20 carbon atoms, less than about 18 carbonatoms, or less than about 16 carbon atoms. In another embodiment, thefatty ester product is a saturated or unsaturated fatty ester producthaving a carbon atom content between 24 and 46 carbon atoms. In oneembodiment, the fatty ester product has a carbon atom content between 24and 32 carbon atoms. In another embodiment, the fatty ester product hasa carbon content of 14 and 20 carbons. In another embodiment, the fattyester is the methyl ester of C_(18:1). In another embodiment, the fattyester is the ethyl ester of C_(16:1). In another embodiment, the fattyester is the methyl ester of C_(16:1). In yet another embodiment, thefatty ester is octadecyl ester of octanol.

Certain microorganisms preferentially produce even- or odd-numberedcarbon chain fatty acids and fatty acid derivatives. For example, E.coli normally produce even-numbered carbon chain fatty acids and fattyacid ethyl esters (FAEE). Surprisingly, the methods disclosed herein canbe used to alter that production. For example, E. coli can be made toproduce odd-numbered carbon chain fatty acids and FAEE under certaincircumstances.

2. Ester Characteristics

An ester typically includes what may be designated an “A” side and a “B”side. The B side may be contributed by a fatty acid produced from denovo synthesis in the production host organism. In some embodiments,where the production host is additionally engineered to make alcohols,including fatty alcohols, the A side is also produced by the productionhost organism. In yet other embodiments, the A side can be provided bythe growth medium. By selecting the desired thioesterase genes, the Bside (and the A side when fatty alcohols are being made) can be designedto be have certain desirable carbon chain characteristics. Thesecharacteristics include, for example, points of branching, points ofunsaturation, and desired carbon chain lengths. Thus, the mutantthioesterases of the invention can be engineered to have alteredproperties, for example, altered specificity and/or increased activity,with regard to preference for accepting certain acyl-CoA or acyl-ACPcompounds as an A side chain as described herein. Accordingly therecombinant cell producing the fatty acid derivatives can be made suchthat it preferentially produces a desired fatty acid derivative productthat is valuable as an end product.

When particular thioesterase genes are selected, the A and B sides willhave similar carbon chain characteristics when they are both contributedby the production host using fatty acid biosynthetic pathwayintermediates. For example, at least about 50%, 60%, 70%, or 80% of thefatty esters produced will have A and B sides that vary by about 2, 4,6, 8, 10, 12, or 14 carbons in length. The A side and the B side canalso display similar branching and saturation levels.

In addition to producing fatty alcohols that contribute to the A side,the production host can produce other short chain alcohols such asethanol, propanol, isopropanol, isobutanol, and butanol forincorporation on the A side using techniques well known in the art. Forexample, butanol can be made by the production host organism. To createbutanol producing cells, the LS9001 strain, for example, can be furtherengineered to express atoB (acetyl-CoA acetyltransferase) fromEscherichia coli K12, β-hydroxybutyryl-CoA dehydrogenase fromButyrivibrio fibrisolvens, crotonase from Clostridium beijerinckii,butyryl CoA dehydrogenase from Clostridium beijerinckii, CoA-acylatingaldehyde dehydrogenase (ALDH) from Cladosporium flavum, and adhEencoding an aldehyde-alcohol dehydrogenase of Clostridium acetobutylicumin the pBAD24 expression vector under the prpBCDE promoter system. Otherproduction host organisms may be similarly modified to produce butanolor other short chain alcohols. For example, ethanol can be produced in aproduction host using the methods described by Kalscheuer et al.,Microbiology, 152:2529-2536, 2006, which is herein incorporated byreference.

III. Genetic Engineering of Production Strain to Increase/Improve FattyAcid Derivative Production/Yield

Heterologous polynucleotide sequences involved in a biosynthetic pathwayfor the production of fatty acid derivatives can be introduced stably ortransiently into a production host cell using techniques known in theart. Non-limiting examples of such techniques include electroporation,calcium phosphate precipitation, DEAE-dextran mediated transfection,liposome-mediated transfection, conjugation, transduction, and genomicintegration. For stable transformation, a DNA sequence can furtherinclude a selectable marker, including, for example, markers forantibiotic resistance, and genes that complement auxotrophicdeficiencies. On the other hand, endogenous polynucleotides involved inthe biosynthetic pathway for the production of fatty acid derivativescan also be mutated using known genomic alteration techniques. Thesestrategies can be applied separately or in combination.

Various embodiments herein utilize an expression vector that includes aheterologous DNA sequence encoding a protein involved in a metabolic orbiosynthetic pathway. Suitable expression vectors include, but are notlimited to, viral vectors (such as baculovirus vectors), phage vectors(such as bacteriophage vectors), plasmids, phagemids, cosmids, fosmids,bacterial artificial chromosomes, viral vectors (e.g., viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus,SV40, herpes simplex virus, and the like), P1-based artificialchromosomes, yeast plasmids, yeast artificial chromosomes, and any othervectors for specific production hosts of interest (such as E. coli,Pseudomonas pisum, and Saccharomyces cerevisiae).

Useful expression vectors can include one or more selectable markergenes to provide a phenotypic trait for selection of transformedproduction host cells. The selectable marker gene encodes a proteinnecessary for the survival or growth of transformed production hostcells grown in a selective culture medium. Production host cells nottransformed with the vector containing the selectable marker gene willnot survive in the culture medium. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins(e.g., ampicillin, neomycin, methotrexate, or tetracycline); (b)complement auxotrophic deficiencies; or (c) supply critical nutrientsnot available from complex media (e.g., the gene that encodes D-alanineracemate for Bacilli). In alternative embodiments, the selectable markergene is one that encodes dihydrofolate reductase or confers neomycinresistance (for use in eukaryotic cell culture), or one that conferstetracycline or ampicillin resistance (for use in a prokaryoticproduction host cell, such as in E. coli).

In the expression vector, the DNA sequence encoding the gene in thebiosynthetic pathway is operably linked to an appropriate expressioncontrol sequence (e.g., promoters, enhancers, and the like) to directsynthesis of the encoded gene product. Such promoters can be derivedfrom microbial or viral sources, including, for example, from CMV andSV40. Depending on the production host/vector system utilized, anynumber of suitable transcription and translation control elements can beused in the expression vector, including constitutive and induciblepromoters, transcription enhancer elements, transcription terminators,and the like. See, e.g., Bitter et al., Methods in Enzymology,153:516-544, 1987.

Suitable promoters for use in prokaryotic production host cells include,but are not limited to, promoters capable of recognizing the T4, T3, Sp6and T7 polymerases, the P_(R) and P_(L) promoters of bacteriophagelambda, the trp, recA, heat shock, and lacZ promoters of E. coli, thealpha-amylase and the sigma-specific promoters of B. subtilis, thepromoters of the bacteriophages of Bacillus, Streptomyces promoters, theint promoter of bacteriophage lambda, the bla promoter of thebeta-lactamase gene of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene. Prokaryotic promoters arereviewed by Glick, J. Indust. Microbiol., 1:277, 1987; Watson et al.,MOLECULAR BIOLOGY OF THE GENE, 4th Ed. (1987), Benjamin Cummins (1987);and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.(Cold Spring Harbor Laboratory Press, 1989), the disclosures of whichare incorporated herein by reference. Non-limiting examples of suitableeukaryotic promoters for use within a eukaryotic production host areviral in origin and include the promoter of the mouse metallothionein Igene (Hamer et al., J. Mol. Appl. Gen., 1:273, 1982); the TK promoter ofherpes virus (McKnight, Cell, 31:355, 1982); the SV40 early promoter(Benoist et al., Nature, 290:304, 1981); the cytomegalovirus promoter(Foecking et al., Gene, 45:101, 1980); the yeast gal4 gene promoter(Johnston et al., PNAS (USA), 79:6971, 1982; Silver et al., PNAS (USA),81:5951, 1984); and the IgG promoter (Orlandi et al., PNAS (USA),86:3833, 1989), the contents of which are incorporated herein byreference.

The production host can be genetically modified with a heterologous genesequence encoding a biosynthetic pathway gene product that is operablylinked to an inducible promoter. Inducible promoters are known in theart. Non-limiting examples of suitable inducible promoters includepromoters that are affected by proteins, metabolites, or chemicals.These include, but are not limited to: a bovine leukemia virus promoter,a metallothionein promoter, a dexamethasone-inducible MMTV promoter, anSV40 promoter, an MRP polIII promoter, a tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter) as well asthose from the trp and lac operons.

In some examples, a production host is genetically modified with aheterologous gene sequence encoding a biosynthetic pathway gene productthat is operably linked to a constitutive promoter. Suitableconstitutive promoters are known in the art and include constitutiveadenovirus major late promoter, a constitutive MPSV promoter, or aconstitutive CMV promoter.

In some examples, a modified production host is one that is geneticallymodified with an exogenous gene sequence encoding a single proteininvolved in a biosynthesis pathway. In other embodiments, a modifiedproduction host is one that is genetically modified with exogenous genesequences encoding two or more proteins involved in a biosynthesispathway, for example, the first and second enzymes in a biosyntheticpathway.

When a production host is genetically modified to express two or moreproteins involved in a biosynthetic pathway, those gene sequences caneach be contained in a single or in separate expression vectors. Whenthose gene sequences are contained in a single expression vector, insome embodiments, the polynucleotide sequences will be operably linkedto a common control element wherein the common control element controlsexpression of all of the biosynthetic pathway protein-encoding genesequences in the single expression vector (e.g., a promoter).

When a modified production host is genetically modified withheterologous DNA sequences encoding two or more proteins involved in abiosynthesis pathway, one of the DNA sequences can be operably linked toan inducible promoter, and one or more of the DNA sequences can beoperably linked to a constitutive promoter.

In some embodiments, the intracellular concentration (i.e., theconcentration within the genetically modified production host) of abiosynthetic pathway intermediate can be increased to further boost theyield of the final product. The intracellular concentration of theintermediate can be increased in a number of ways, including, but notlimited to, increasing the concentration in the culture medium of asubstrate for a biosynthetic pathway; increasing the catalytic activityof an enzyme that is active in the biosynthetic pathway; increasing theintracellular amount of a substrate (e.g., a primary substrate) for anenzyme that is active in the biosynthetic pathway; and the like.

In some examples, the fatty acid derivative or intermediate is producedin the cytoplasm of the production host. The cytoplasmic concentrationcan be increased in a number of ways, including, but not limited to,binding of the fatty acid to coenzyme A to form an acyl-CoA thioester.Additionally, the concentration of acyl-CoA can be increased byincreasing the biosynthesis of CoA in the cell, such as byover-expressing genes associated with pantothenate biosynthesis (e.g.,panD) or knocking out genes associated with glutathione biosynthesis(e.g., glutathione synthase).

Regulatory sequences, coding sequences, and combinations thereof, can beintroduced or altered in the chromosome of the production host. In someexamples, the integration of the desired recombinant sequence into theproduction host genomic sequence does not require the use of aselectable marker such as an antibiotic. In some examples, the genomicalterations include changing the control sequence of the target genes byreplacing the native promoter(s) with a promoter that is insensitive toregulation. There are numerous approaches for doing this. For example,Valle and Flores, in Methods Mol. Biol., 267:113-122, 2006, describe aPCR-based method to overexpress chromosomal genes in E. coli. Thecontent of Valle and Flores is incorporated by reference herein. Anotherapproach is based on the use of single-stranded oligonucleotides tocreate specific mutations directly in the chromosome, using thetechnique developed by Court et al., PNAS(USA), 100:15748-15753, 2003,the content of which is also incorporated herein by reference. Thistechnique is based on the use of the overexpression of the Beta proteinfrom the bacteriophage lambda to enhance genetic recombination. Theadvantages of this approach include that synthetic oligonucleotides 70bases long (or more) can be used to create point mutations, insertions,and deletions, thus eliminating any cloning steps. Furthermore, thesystem is sufficiently efficient that no markers are necessary toisolate the desired mutations.

With this approach the regulatory region of a gene can be changed tocreate a stronger promoter and/or eliminate the binding site of arepressor. Accordingly, a desired gene can be overexpressed in theproduction host organism.

IV. Fermentation

A. Maximizing Production Efficiency

Production and isolation of fatty acid derivatives can be enhanced byemploying specific fermentation techniques. One method for maximizingproduction while reducing costs is increasing the percentage of thecarbon source that is converted to hydrocarbon products.

During normal cellular lifecycles, carbon is used in cellular functionsto produce lipids, saccharides, proteins, organic acids, andpolynucleotides. Reducing the amount of carbon necessary forgrowth-related activities can increase the efficiency of carbon sourceconversion to output. This can be achieved by first growingmicroorganisms to a desired density, which is achieved at the peak ofthe growth log phase. Then, replication checkpoint genes can beharnessed to stop the growth of cells. Specifically, quorum sensingmechanisms (as reviewed in Camilli and Bassler, Science, 311:1113, 2006;Venturi, FEMS Microbio. Rev., 30:274-291, 2006; and Reading andSperandio, FEMS Microbiol. Lett., 254:1-11, 2006, the disclosures ofwhich are incorporated by reference herein) can be used to activategenes associated with the stationary phase.

Genes that can be activated to stop cell replication and growth in E.coli include umuDC genes, the over-expression of which stops theprogression from stationary phase to exponential growth (Murli et al.,J. of Bact., 182:1127, 2000). UmuC is a DNA polymerase that can carryout translesion synthesis over non-coding lesions—the mechanistic basisof most UV and chemical mutagenesis. The umuDC gene products are usedfor the process of translesion synthesis and also serve aspolynucleotide sequence damage checkpoints. The umuDC gene productsinclude UmuC, UmuD, umuD′, UmuD′₂C, UmuD′₂, and/or UmuD₂. In the meantime, the product-producing genes can be activated, thus minimizing theneed for replication and maintenance pathways to be used while the fattyacid derivative is being made. Production host microorganisms can alsobe engineered to express umuC and/or umuD from E. coli in pBAD24 underthe prpBCDE promoter system through de novo synthesis of these geneswith the appropriate end-product production genes.

The percentage of input carbons converted to fatty esters or hydrocarbonproducts is a cost driver. The more efficient the process is (i.e., thehigher the percentage of input carbons converted to fatty esters orhydrocarbon products), the less expensive the process is. Foroxygen-containing carbon sources (e.g., glucose and other carbohydratebased sources), the oxygen is released in the form of carbon dioxide.For every 2 oxygen atoms released, a carbon atom is also released,leading to a maximal theoretical metabolic efficiency of about 34% (w/w)(for fatty acid derived products). This figure, however, changes forother hydrocarbon products and carbon sources. Typical efficiencies inthe literature are about <5%. Production hosts engineered to producehydrocarbon products can have greater than about 1%, for example,greater than about 3%, 5%, 10%, 15%, 20%, 25%, or 30% efficiency. In oneexample, production hosts will exhibit an efficiency of about 10% toabout 25%. In other examples, such production hosts will exhibit anefficiency of about 25% to about 30%. In other examples, such productionhosts will exhibit >30% efficiency.

The production host can be additionally engineered to expressrecombinant cellulosomes, such as those described in PCT applicationnumber PCT/US2007/003736, incorporated herein by reference in itsentirety, which can allow the production host to use cellulosic materialas a carbon source. For example, the production host can be additionallyengineered to express invertases (EC 3.2.1.26) so that sucrose can beused as a carbon source.

Similarly, the production host can be engineered using the teachingsdescribed in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846;and 5,602,030, all incorporated herein by reference in their entirety,so that the production host can assimilate carbon efficiently and usecellulosic materials as carbon sources.

In one example, the fermentation chamber encloses a fermentationrun/mixture that is undergoing a continuous reduction. In this instance,a stable reductive environment is created. The electron balance ismaintained by the release of carbon dioxide (in gaseous form). Effortsto augment the NAD/H and NADP/H balance can also facilitate instabilizing the electron balance.

The availability of intracellular NADPH can also be enhanced byengineering the production host to express an NADH:NADPHtranshydrogenase. The expression of one or more NADH:NADPHtranshydrogenases converts the NADH produced in glycolysis to NADPHwhich enhances the production of fatty acid derivatives.

B. Small-Scale Hydrocarbon Production

For small scale hydrocarbon product production, E. coli BL21(DE3) cellsharboring pBAD24 (with ampicillin resistance and the end-productsynthesis pathway) as well as pUMVC1 (with kanamycin resistance and theacetyl CoA/malonyl CoA over-expression system) are incubated overnightin 2 Liter flasks at 37° C., shaken at >200 rpm in 500 mL LB mediumsupplemented with 75 μg/mL ampicillin and 50 μg/mL kanamycin until thecultures reach an OD₆₀₀ of >0.8. Upon achieving an OD₆₀₀ of >0.8, cellsare supplemented with 25 mM sodium propionate (at pH 8.0) to activatethe engineered gene systems for production, and to stop cellularproliferation by activating UmuC and UmuD proteins. The induction stepis performed for 6 hours at 30° C. After incubation, the medium isexamined for hydrocarbon product using GC-MS.

C. Large-Scale Hydrocarbon Production

For large scale product production, the engineered production hosts aregrown in batches of 10 Liter, 100 Liter, or larger; fermented; andinduced to express the desired products based on the specific genesencoded in the appropriate plasmids therein.

For example, E. coli BL21(DE3) cells harboring pBAD24 (with ampicillinresistance and the end-product synthesis pathway) as well as pUMVC1(with kanamycin resistance and the acetyl-CoA/malonyl-CoAoverexpression) are incubated from a 500-mL seed culture for a 10-Literfermentation run (or a 5-Liter seed culture for a 100-Literfermentation) in an LB medium (glycerol free) containing 50 μg/mLkanamycin and 75 μg/mL ampicillin at 37° C., which is shaken at >200 rpmuntil the culture reaches an OD₆₀₀ of >0.8, a process that typicallytakes about 16 hours. The fermentation medium is continuouslysupplemented so as to maintain a sodium phosphate of 25 mM, at pH 8.0,in order to activate the engineered gene systems for production, and tostop cellular proliferation by activating UmuC and UmuD proteins. Themedium is also continuously supplemented with glucose to maintain aconcentration of 25 g/100 mL.

After the first hour of induction, an aliquot of no more than 10% of thetotal cell volume is removed each hour and allowed to settle withoutagitation, which in turn allows the hydrocarbon product(s) to rise tothe surface, undergoing a spontaneous phase separation. The hydrocarboncomponent is collected and the aqueous phase returned to the reactionchamber. The reaction chamber is operated continuously. When the OD₆₀₀drops below about 0.6, the cells are replaced with a new batch grownfrom a seed culture.

For wax ester production, the wax esters are isolated, washed briefly in1 M HCl, and returned to pH 7 through extensive washing with distilledwater.

V. Post-Production Processing

The fatty acid derivatives produced during fermentation can be separatedfrom the fermentation media. Any technique known for separating fattyacid derivatives from aqueous media can be used. An exemplary separationprocess is a two-phase (bi-phasic) separation process. This processinvolves fermenting the genetically engineered production hosts underconditions sufficient to produce a fatty acid derivative, allowing thederivative to collect in an organic phase, and separating the organicphase from the aqueous fermentation broth. This method can be practicedin both a batch and continuous fermentation setting.

Bi-phasic separation takes advantage of the relative immiscibility offatty acid derivatives to facilitate separation. “Immiscibility” refersto the relative inability of a compound to dissolve in water and isdefined and/or determined by the compounds partition coefficient. One orordinary skill in the art will appreciate that by choosing afermentation broth and organic phase such that the fatty acid derivativebeing produced has a high logP value, the fatty acid derivative willseparate into the organic phase in the fermentation vessel, even at lowconcentrations.

The fatty acid derivatives produced in accordance to the compositions,vectors, cells, and methods herein will be relatively immiscible in thefermentation broth, as well as in the cytoplasm. Therefore, the fattyacid derivative will collect in an organic phase either intracellularlyand/or extracellularly. The collection of the products in the organicphase will lessen the impact of the fatty acid derivatives on cellularfunction, and will allow the production host to produce greater amountof product for longer.

The fatty alcohols, fatty esters, waxes, and hydrocarbons produced inaccordance to the disclosures herein allow for the production ofhomogeneous compounds wherein at least about 60%, 70%, 80%, 90%, 91%,92%, 93%, 94%, or 95% of the fatty alcohols, fatty esters, and waxesproduced suitably have carbon chain lengths that vary by less than about6, less than about 4 carbons, or less than about 2 carbons. Thesecompounds can also be produced so that they have a relatively uniformdegree of saturation, for example, at least about 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, or 95% of the fatty alcohols, fatty esters,hydrocarbons and waxes are monounsaturated, diunsaturated, ortriunsaturated. These compounds can be used directly as products orcomponents of products, for example, as fuels, detergents, lubricants,personal care additives, nutritional supplements etc. These compoundscan also be used as feedstock for subsequent reactions to make otherproducts, including, for example transesterification, hydrogenation,catalytic cracking (via hydrogenation, pyrolysis, or both), orepoxidation reactions.

The fatty alcohols, fatty esters, waxes, and hydrocarbons produced inaccordance to the compositions, vectors, cells, and methods hereincontain low levels of unwanted or undesired elements, including, but notlimited to, heavy metals. In some embodiments, the fatty alcohols, fattyesters, waxes, and hydrocarbons produced as described herein suitablycontain less than about 50 ppm arsenic; less than about 300 ppm calcium;less than about 200 ppm chlorine; less than about 50 ppm cobalt; lessthan about 50 ppm copper; less than about 300 ppm iron; less than about2% by weight of water; less than about 50 ppm lead; less than about 50ppm manganese; less than about 0.2 ppm mercury; less than about 50 ppmmolybdenum; less than about 1% by weight of nitrogen; less than about200 ppm potassium; less than about 300 ppm sodium; less than about 3% byweight of sulfur; less than 50 ppm zinc; and/or less than 700 ppmphosphorus.

In some embodiments, the fatty alcohols, fatty esters, waxes, andhydrocarbons produced in accordance to the disclosures herein containbetween about 50% and about 90% carbon; between about 5% and about 25%hydrogen; or between about 5% and about 25% oxygen. In otherembodiments, the fatty alcohols, fatty esters, waxes, and hydrocarbonsproduced as described herein contain between about 65% and about 85%carbon; between about 10% and about 15% hydrogen; or between about 10%and about 20% oxygen.

VI. Fuel Compositions

As provided herein, certain fatty acid derivatives made according to themethods and compositions described herein possess various advantageouscharacteristics for use as a fuel. One of ordinary skill in the art willappreciate that, depending upon the intended purpose of the fuel,different fatty acid derivatives may have advantages as compared toothers fatty acid derivatives. For example, branched fatty acidderivatives may be more desirable as automobile fuels or components ofautomobile fuels that are intended for uses in cold climates. Similarly,for certain applications, it may be advantageous to produce a fuel thatis either more or less oxygenated or more or less saturated.

Using the methods described herein, fuels comprising relativelyhomogeneous fatty acid derivatives that at the same time have thedesired characteristics/qualities can be produced. Such fatty acidderivative-based fuels can be characterized by carbon fingerprinting,and their lack of impurities, when compared to petroleum derived fuelsor biodiesel derived from triglyceride, is also advantageous. The fattyacid derivative-based fuels can be combined with other fuels or fueladditives to produce fuels having desired properties.

The production hosts and methods disclosed herein can be used to producefree fatty acids and fatty esters. In some embodiments, the productionhosts and methods disclosed herein can be used to produce a higherand/or improved titer or yield of fatty acid derivatives, including, forexample, free fatty acids and/or fatty esters. In some embodiments, thepercentage of free fatty acids in the product produced by the productionhost is at least about 1%, for example, at least about 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In some embodiments, thepercentage of fatty esters in the product produced by the productionhost is at least about 50%, for example, at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, or 90%. In some embodiments, the ratio of fattyesters to free fatty acids in the product produced by the productionhost is about 10:1, 9:1, 8:1, 7:1, 5:1, 2:1, or 1:1. In certainembodiments, the fatty ester produced by the production host is ethyldodecanoate, ethyl tridecanoate, ethyl tetradecanoate, ethylpentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate, ethylheptadecanoate, ethyl cis-11-octadecenoate, ethyl octadecanoate, orcombinations thereof. In certain other embodiments, the fatty esterproduced by the production is methyl dedecanoate, methyl tridecanoate,methyl tetradecanoate, methyl pentadecanoate, methylcis-9-hexadecenoate, methyl hexadecanoate, methyl heptadecanoate, methylcis-11-octadecenoate, methyl octadecanoate, or combinations thereof. Incertain embodiments, the free fatty acid produced by the production hostis dodecanoic acid, tetradecanoic acid, pentadecanoic acid,cis-9-hexadecenoic acid, hexadecanoic acid, cis-11-octadecenoic acid, orcombinations thereof.

The production hosts and methods disclosed herein can be used to producedifferent proportions of free fatty acids and fatty esters. In someembodiments, the proportion of free fatty acids in the product can bemodified according to the methods, compositions, vectors and cellsdescribed herein such that the proportion is higher or lower vs. thefatty esters that are produced. In certain related embodiments, theproportion of fatty esters in the product can also be modified accordingto the disclosures herein, such that the proportion is higher or lowervs. the other products, for example, the free fatty acids, that areproduced. In certain other embodiments, the proportional yield of fattyacid derivative with certain carbon chain lengths can be increased ordecreased.

A. Carbon Fingerprinting

Biologically produced fatty acid derivatives represent a new source offuels, such as alcohols, diesel, and gasoline. Biofuels made accordingto the methods and compositions described herein have not heretoforebeen produced from renewable sources and are new compositions of matter.These new fuels can be distinguished from fuels derived frompetrochemical carbon on the basis of dual carbon-isotopicfingerprinting. Additionally, the specific source of biosourced carbon(e.g., glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see U.S. Pat. No. 7,169,588, which is hereinincorporated by reference in its entirety, in particular, at col. 4,line 31, to col. 6, line 8).

The fatty acid derivatives and the associated biofuels, chemicals, andmixtures can be distinguished from their petrochemical derivedcounterparts on the basis of ¹⁴C (f_(M)) and dual carbon-isotopicfingerprinting.

The fatty acid derivatives described herein have utility in theproduction of biofuels and chemicals. The new fatty acidderivative-based products provided by the instant invention additionallycan be distinguished on the basis of dual carbon-isotopic fingerprintingfrom those materials derived solely from petrochemical sources. Theability to distinguish these products is beneficial in tracking thesematerials in commerce. For example, fuels or chemicals comprising both“new” and “old” carbon isotope profiles can be distinguished from fuelsand chemicals made only of “old” materials. Thus, the instant materialscan be followed or “tracked” in commerce or identified in commerce as abiofuel on the basis of their unique profile. In addition, othercompeting materials can be identified as being biologically derived orderived from a petrochemical source.

In some examples, a biofuel composition is made, which includes a fattyacid derivative having δ¹³C of from about −10.9 to about −15.4, whereinthe fatty acid derivative accounts for at least about 85% of biosourcedmaterial (i.e., derived from a renewable resource such as, for example,cellulosic materials and sugars) in the composition. In other examples,the biofuel composition includes a fatty acid derivative having theformula:

X—(CH(R))_(n)CH₃

wherein

-   -   X=CH₃, —CH₂OR¹; —C(O)OR²; or —C(O)NR³R⁴;    -   R=for each n, independently absent, an H, or a lower aliphatic;    -   n=an integer from about 8 to about 34, preferably an integer        from about 10 to about 24;

R¹, R², R³, R⁴=independently selected from an H or a lower alkyl.

Typically, when R is a lower aliphatic group, R represents a branched,unbranched or cyclic lower alkyl or lower alkenyl moiety. Exemplary Rgroups include, without limitation, methyl, isopropyl, isobutyl,sec-butyl, cyclopentenyl, and the like. The fatty acid derivative isadditionally characterized as having a δ¹³C of from about −10.9 to about−15.4, and the fatty acid derivative accounts for at least about 85% ofbiosourced material in the composition. In some examples the fatty acidderivative in the biofuel composition is characterized by having afraction of modern carbon (f_(M) ¹⁴C) of at least about 1.003, 1.010, or1.5.

B. Impurities

The fatty acid derivatives prepared in accordance with the disclosuresherein are useful as components of or for making biofuels as well asother industrial chemicals. These fatty acid derivatives are madedirectly from fatty acids and not from the chemical processing oftriglycerides. Accordingly, fuels and other industrial chemicalscomprising the disclosed fatty acid derivatives often contain fewerimpurities than are normally associated with, for example, productsderived from triglycerides such as fuels derived from vegetable oils andfats.

The crude fatty acid derivative biofuels prepared in accordance with thedisclosures herein (prior to mixing the fatty acid derivative with otherfuels such as petroleum-based fuels) contain less transesterificationcatalysts than petroleum-based diesel or other biodiesel produced viaone or more transesterification steps. The fatty acid derivative cancontain less than about 2.0%, for example, less than about 1.5%, 1.0%,0.5%, 0.3%, 0.1%, 0.05%, or 0% of a transesterification catalyst or animpurity resulting from a transesterification catalyst. Non-limitingexamples of transesterification catalysts include hydroxide catalysts,such as NaOH, KOH, and LiOH; and acidic catalysts, such as mineral acidcatalysts and Lewis acid catalysts. Non-limiting examples of catalystsand impurities resulting from transesterification catalysts include tin,lead, mercury, cadmium, zinc, titanium, zirconium, hafnium, boron,aluminum, phosphorus, arsenic, antimony, bismuth, calcium, magnesium,strontium, uranium, potassium, sodium, lithium, and combinationsthereof.

The crude fatty acid derivative biofuels prepared in accordance with thedisclosures herein (prior to mixing the fatty acid derivatives with oneor more other fuels) tend to have a low gelling point, especially whenthe fatty acid derivative product comprises a Ciba ethyl ester or a Cibaethyl ester, as compared to the gelling points of other types ofbiofuels.

Similarly, the crude fatty acid derivative biofuels prepared inaccordance with the disclosures herein (prior to mixing the fatty acidderivative(s) with one or more other fuels such as petroleum-baseddiesels or other biodiesels) contain less glycerol (or glycerin) thanbiofuels made from triglycerides. The fatty acid derivative(s) cancontain less than about 2.0%, for example, less than about 1.5%, 1.0%,0.5%, 0.3%, 0.1%, 0.05%, or 0% by weight of glycerol.

Crude biofuels derived from the fatty acid derivatives herein alsocontain less free alcohol(s) (e.g., alcohols that are used to create theester) than biodiesels made from triglycerides. This is due in part tothe efficiency of utilization of the alcohols by the production hosts ofthe present disclosure. For example, the fatty acid derivative(s) cancontain less than about 2.0%, 1.5%, 1.0%, 0.5%, 0.3%, 0.1%, 0.05%, or 0%by weight of free alcohol.

Biofuel derived from the disclosed fatty acid derivatives can beadditionally characterized by its low concentration of sulfur ascompared to petroleum-derived diesel. Biofuel derived from fatty acidderivatives herein can have less than about 2.0%, for example, less thanabout 1.5%, 1.0%, 0.5%, 0.3%, 0.1%, 0.05%, or 0% by weight of sulfur.

C. Additives and Fuel Compositions

Fuel additives are used to enhance the performance of a fuel or engine.For example, fuel additives can be used to alter the freezing/gellingpoints, cloud points, lubricity, viscosity, oxidative stability,ignition quality, octane levels, and flash points. In the United States,all fuel additives must be registered with Environmental ProtectionAgency. The names of fuel additives and the companies that sell the fueladditives are publicly available by contacting the EPA or by viewing theagency's website. One of ordinary skill in the art will appreciate thatthe fatty acid derivatives described herein can be mixed with one ormore fuel additives to impart a desired quality.

The fatty acid derivatives described herein can be formulated intosuitable fuel additives, which enhances the performance of fuels orengines. For example, the fatty acid derivatives described herein can beformulated into lubricity improvers, which impart desirable propertiessuch as wear protection to the engine parts. Accordingly, additivecompositions comprising the fatty acid derivatives produced inaccordance with the disclosures herein are provided. In another example,the fatty acid derivatives described herein can be formulated intocorrosion inhibitors.

The fatty acid derivatives described herein can be mixed with otherfuels such as one or more biodiesels derived from triglycerides, variousalcohols such as ethanol and butanol, and petroleum-derived productssuch as gasoline or diesel. Under certain circumstances, a fatty acidderivative with a low gelling point, such as a C_(16:1) ethyl ester or aC_(18:1) ethyl ester, is produced. This low gelling point fatty acidderivative can be mixed with one or more biodiesels made fromtriglycerides to reduce gelling point of the resulting fuel whencompared to a fuel containing only the one or more biodiesels made fromtriglycerides. Similarly, a fatty acid derivative, such as a C_(16:1)ethyl ester or a C_(18:1) ethyl ester, can be mixed with apetroleum-derived diesel to provide a mixture that contains at leastabout, and often greater than about, 5% by weight of biodiesel. In someexamples, the fuel mixture includes at least about 10%, 15%, 20%, 30%,40%, 50%, and 60% by weight of the fatty acid derivative.

In some embodiments, the fuel composition can further comprise asynthetic fuel. Any synthetic fuel obtained from coal, natural gas, orbiomass can be suitably used. In a further embodiments, the syntheticfuel comprises a Fischer-Tropsch based fuel, a Bergius-based fuel, aMobil-based fuel, a Karrick-based fuel, or a combination thereof. Instill further embodiments, the synthetic fuel comprises aCoal-To-Liquids based fuel (CTL-based fuel), a Gas-To-Liquids based fuel(GTL-based fuel), a Biomass-To-Liquids based fuel (BTL-based fuel), aCoal and Biomass-To-Liquids based fuel (CBTL-based fuel), or acombination thereof. In an exemplary embodiment, the synthetic fuelcomprises a Fischer-Tropsch-based fuel.

The amount of synthetic fuel in the fuel composition disclosed hereinmay be from about 5% to about 90%, from about 5% to about 80%, fromabout 5% to about 70%, from about 5% to about 60%, or from about 5% toabout 50%.

In certain embodiments, a biofuel composition can be made that includesat least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or95% of a fatty acid derivative that includes a carbon chain that is 8:0,10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1,20:2, 20:3, 22:0, 22:1 or 22:3. Such biofuel compositions canadditionally include at least one additive selected from a cloud pointlowering additive that can lower the cloud point to less than about 5°C., or less than about 0° C.; a surfactant; a microemulsion; at leastabout 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95%diesel fuel from triglycerides; a petroleum-derived gasoline; or adiesel fuel from petroleum.

In some embodiments, the fuel composition comprising the fatty estersproduced in accordance with the methods, vectors, cells and compositionsherein further comprises one or more diesel fuel additives. Suitableadditives are desirably those that afford improved performance but alsocompatibility with the components in the fuel composition and devicesthat are typically associated with diesel engines. Illustrative examplesof other suitable fuel additives include ignition improvers or cetanenumber improvers, detergents, dispersants, antiwear agents, viscosityindex modifiers, friction modifiers, lubricity improvers, stabilizers,antioxidants, corrosion inhibitors, biocides, metal deactivators, andminor amounts of other optional additives, including, withoutlimitation, antifoaming agents and seal fixes.

In particular embodiments, ignition improvers or cetane number improversare often added to improve diesel engine performance. Exemplary cetanenumber improvers include 2′-ethylhexyl nitrate, and other alkylnitrates. Cetane number improvers can be added to a fuel composition inan amount that is about 0.01 wt. % to about 1.0 wt. %, for example,about 0.05 wt. % to about 0.5 wt. %, based on the total weight of thefuel composition.

In certain embodiments, various detergents and/or dispersants can beincluded in the fuel composition comprising the fatty ester produced inaccordance with the present disclosures to associate and disperse orremove harmful deposits from diesel engine parts. Suitable detergentstypically comprise a polar head comprising a metal salt of an acidicorganic compound and a long hydrophobic tail. Exemplary detergentsinclude borated carbonate salts, borated sulfonate salts, which arepreferably overbased. See, e.g., U.S. Pat. Nos. 4,744,920, 4,965,003,the disclosures of which are incorporated herein. Exemplary dispersantsinclude, without limitation, carboxylic dispersants, succinimidedispersants, amine dispersants, and Mannich dispersants. See, e.g., U.S.Pat. Nos. 3,172,892, 3,438,757, 3,980,569, and 6,165,235, thedisclosures of which are incorporated by reference herein. Dispersantscan be present in the fuel composition in an amount of about 0.01 wt. %to about 0.1 wt. %, for example, 0.03 to about 0.05 wt. %, based on thetotal weight of the fuel composition.

In certain embodiments, antiwear agents, including for example,dihydrocarbyl dithiophosphate metal salts, can be added to the fuelcomposition to provide both antiwear and antioxidation benefits. See,e.g., U.S. Pat. No. 5,898,023, the disclosures of which are incorporatedherein by reference.

In particular embodiments, the amount of lubricity improver in the fuelcomposition can range from about 1 ppm to about 50,000 ppm, for example,about 10 ppm to about 20,000 ppm, or about 25 ppm to about 10,000 ppm.Non-limiting examples of lubricity improvers include esters and fattyacids, which may or may not be the same as those produced in accordanceto the methods described herein.

In particular embodiments, the amount of stabilizers, which improves thestorage stability of the fuel composition, can range from about 0.001wt. % to about 2 wt. %, for example about 0.01 wt. % to about 1 wt. %,based on the total weight of the fuel composition. An exemplarystabilizer is a tertiary alkyl primary amine.

Antioxidants prevent the formation of gum depositions on fuel systemcomponents due to oxidation of the fuels in storage and/or inhibit theformation of peroxide compounds in certain fuel compositions. The amountof antioxidants can be ranged from about 0.001 wt. % to about 5 wt. %,for example, from about 0.01 wt. % to about 1 wt. %, based on the totalweight of the fuel composition.

Corrosion inhibitors protect ferrous metals in fuel handling systems,such as pipelines and storage tanks, from corrosion. Certain corrosioninhibitors are also known to impart additional lubricity, and as suchare particularly suitable when additional lubricity is desired. Thecorrosion inhibitor may be present in the fuel composition in an amountof about 0.001 wt. % to about 5 wt. %, for example, from about 0.01 wt.% to about 1 wt. %, based on the total weight of the fuel composition.

Biocides are used to combat microbial growth in the fuel composition,which may be present in the fuel composition at a concentration of about0.001 wt. % to about 5 wt. %, for example, from about 0.01 wt. % toabout 1 wt. %, based on the total weight of the fuel composition.

Metal deactivators suppress the catalytic effects of some metals,particularly copper, have on fuel oxidation, which can be present in thefuel composition in an amount of about 0.001 wt. % to about 5 wt. %, forexample, at 0.01 wt. % to about 1 wt. %, based on the total weight ofthe fuel composition.

In addition, viscosity improvers, which are typically polymericmaterials of number average molecular weights of from about 5,000 toabout 250,000, and friction modifiers, which are typicallysulfur-containing organo-molybdenum compounds can be added in minoramounts. Foam inhibitors, which typically include alkyl methacrylatepolymers or dimethyl silicon polymers, can also be added to the fuelcomposition in an amount of less than about 10 ppm. Furthermore, sealfixes can be added to insure proper elastomer sealing and preventpremature seal failure can be included in the fuel composition.

EXAMPLES

The examples that follow illustrate the engineering of production hoststo produce specific fatty acid derivatives. The biosynthetic pathwaysinvolved in the production of fatty acid derivatives are illustrated inthe figures.

For example, FIG. 3 is a diagram of the FAS pathway depicting theenzymes directly involved in the synthesis of acyl-ACP. To increase theproduction of fatty acid derivatives, such as waxes, fatty esters, fattyalcohols, and hydrocarbons, one or more of the enzymes described thereincan be over expressed or mutated to reduce feedback inhibition, in orderto increase the amount of acyl-ACP produced. Additionally, enzymes thatmetabolize the intermediates to make non-fatty acid based products(e.g., side reactions) can be functionally deleted or attenuated toincrease the flux of carbon through the fatty acid biosynthetic (FAS)pathway. In the examples below, many production hosts are described thathave been modified to increase fatty acid production.

FIGS. 4 and 5 depict biosynthetic pathways that can be engineered tomake fatty esters and fatty alcohols, respectively. The conversion ofeach substrate (e.g., acetyl-CoA, malonyl-CoA, acyl-ACP, fatty acid, andacyl-CoA) to each product (e.g., acetyl-CoA, malonyl-CoA, acyl-ACP,fatty acid, acyl-CoA, fatty aldehydes, fatty esters, and fatty alcohols)can be accomplished using several different polypeptides that aremembers of the enzyme classes indicated.

The examples below describe microorganisms that have been engineered orcan be engineered to produce specific fatty alcohols, fatty esters, andhydrocarbons.

Example 1. Production Host Construction

An exemplary production host is LS9001. LS9001 was produced by modifyingC41(DE3) from Overexpress (Saint Beausine, France) to knockout the fadEgene (acyl-CoA dehydrogenase).

Briefly, the fadE knockout strain of E. coli was prepared using primersYafV_NotI and Ivry_O1 to amplify about 830 bp upstream of fadE andprimers Lpcaf of and LpcaR_Bam to amplify about 960 bp downstream offadE. Overlap PCR was used to create a construct for in-frame deletionof the complete fadE gene. The fadE deletion construct was cloned intothe temperature-sensitive plasmid pKOV3, which contained a sacB gene forcounterselection, and a chromosomal deletion of fadE was made accordingto the method of Link et al., J. Bact. 179:6228-6237, 1997. Theresulting strain was not capable of degrading fatty acids and fattyacyl-COAs. This knockout strain is herein designated as E. coli (DE3,ΔfadE).

Another fadE deletion strain, MG1655, was construted according to theprocedures described by Datsenko et al., PNAS(USA), 97:6640-6645 (2000),with the modifications described below. The two primers used to createthe deletion were:

Del-fadE-F: (SEQ ID NO: 69)5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATATTGATTCCGGGGATCCGTCGACC; and Del-fadE-R: (SEQ ID NO: 70)5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTTCCTGTAGGCTGGAGCTGCTTC.

The Del-fadE-F and Del-fadE-R primers each contain 50 bases of homologyto the E. coli fadE gene and were used to amplify the Kanamycinresistance cassette from plasmid pKD13 by PCT as described. Theresulting PCR product was used to transform electrocompetent E. coliMG1655 cells containing pKD46. The cells were previously induced witharabinose for 3-4 hours as described by Datsenko, supra. Following 3hours of outgrowth in an SOC medium at 37° C., the cells were plated onLuria agar plates containing 50 μg/mL of Kanamycin. Resistant colonieswere isolated after an overnight incubation at 37° C. Disruption of thefadE gene was confirmed in some of the colonies by PCR amplication usingprimers fadE-L2 and fadE-R1, which were designed to flank the fadE gene.

fadE-L2 (SEQ ID NO: 71) 5′-CGGGCAGGTGCTATGACCAGGAC; and fadE-R1(SEQ ID NO: 72) 5′-CGCGGCGTTGACCGGCAGCCTGG

After the proper fadE deletion was confirmed, one colony was used toremove the Km^(R) marker using the pCP20 plasmid. The resulting strainis designaed as MG1655 (ΔfadE).

The fadE-deleted hosts were subject to further adjustments. A plasmidcarrying the four genes that are responsible for acetyl-CoA carboxylaseactivity in E. coli (accA, accB, accC, and accD, GenBank Accession Nos:NP_414727, NP_417721, NP_417722, NP_416819, EC 6.4.1.2) were introduced.The accABCD genes were cloned in two steps as bicistronic operons intothe NcoI/HindIII and NdeI/AvrII sites of pACYCDuet-1 (Novagen, Madison,Wis.), and the resulting plasmid was designated as pAS004.126.Alternatively, the production host was engineered to express accABCDfrom Lactobacillus plantarum.

Additional modifications that were included in a production hostincluded the following: overexpression of aceEF (encoding the Elpdehydrogenase component and the E2p dihydrolipoamide acyltransferasecomponent of the pyruvate and 2-oxoglutarate dehydrogenase complexes);and fabH/fabD/fabG/acpP/fabF (encoding FAS) from E. coli, Nitrosomonaseuropaea (ATCC 19718), Bacillus subtilis, Saccharomyces cerevisiae,Streptomyces spp, Ralstonia, Rhodococcus, Corynebacteria, Brevibacteria,Mycobacteria, and oleaginous yeast. Similarly, production hosts wereengineered to express accABCD (encoding acetyl CoA carboxylase) fromPisum savitum. However, when the production host was also producingbutanol it was found less desirable to express the Pisum savitumhomolog.

In some production hosts, genes were knocked out or attenuated using themethod of Link, et al., J. Bacteriol. 179:6228-6237, 1997. Genes thatwere knocked out or attenuated included gpsA (encoding biosyntheticsn-glycerol 3-phosphate dehydrogenase, GenBank Accession No. NP_418065,EC: 1.1.1.94); ldhA (encoding lactate dehydrogenase, GenBank AccessionNo. NP_415898, EC: 1.1.1.28); pflb (encoding formate acetyltransferase1, GenBank Accession No. P09373, EC: 2.3.1.54); adhE (encoding alcoholdehydrogenase, GenBank Accession No. CAA47743, EC: 1.1.1.1, 1.2.1.10);pta (encoding phosphotransacetylase, GenBank Accession No. NP_416800,EC: 2.3.1.8); poxB (encoding pyruvate oxidase, GenBank Accession No.NP_415392, EC: 1.2.2.2); ackA (encoding acetate kinase, GenBankAccession No. NP_416799, EC: 2.7.2.1), and combinations thereof.

Similarly, the PlsB[D311E] mutation was introduced into LS9001 toattenuate plsB for the fadE deletion. This mutation decreased the amountof carbon diverted to phospholipid production. An allele encodingPlsB[D311E] was made by replacing the GAC codon for aspartate 311 with aGAA codon for glutamate. The altered allele was prepared by genesynthesis and the chromosomal plsB wildtype allele was exchanged for themutant plsB [D311E] allele using the method of Link et al. (see supra).

Example 2. Production Host Modifications

The following plasmids were constructed for the expression of variousproteins that are used in the synthesis of fatty acid derivatives. Theconstructs were prepared using standard molecular biology methods. Allthe cloned genes were put under the control of IPTG-inducible promoters(e.g., a T7 promoter, a tac promoter, or a lac promoter).

The 'tesA gene (thioesterase A gene, GenBank Accession No. NP_415027without leader sequence (SEQ ID NO:31) (Cho and Cronan, J. Biol. Chem.,270:4216-9, 1995, EC: 3.1.1.5, 3.1.2.−)) of E. coli was cloned into anNdeI/AvrII digested pETDuet-1 vector (pETDuet-1 described herein isavailable from Novagen, Madison, Wis.). Genes encoding FatB-type plantthioesterases (TEs) from Umbellularia californica, Cuphea hookeriana,and Cinnamonum camphorum (GenBank Accession Nos: UcFatB1=AAA34215,ChFatB2=AAC49269, ChFatB3=AAC72881, CcFatB=AAC49151) were individuallycloned into three different vectors: (i) NdeI/AvrII digested pETDuet-1;(ii) XhoI/HindIII digested pBluescript KS+(Stratagene, La Jolla, Calif.,to create N-terminal lacZ::TE fusion proteins); and (iii) XbaI/HindIIIdigested pMAL-c2X (New England Lab, Ipswich, Mass.) (to createn-terminal malE::TE fusions). The fadD gene (encoding acyl-CoA synthase)from E. coli was cloned into a NcoI/HindIII digested pCDFDuet-1derivative, which contained the acr1 gene (acyl-CoA reductase) fromAcinetobacter baylyi ADP1 within its NdeI/AvrII sites.

Table 7 provides a summary of the plasmids generated to make severalexemplary production hosts.

TABLE 7 Summary of plasmids used in production hosts GenBank AccessionSource Organism No. & EC Plasmid Gene Product number pETDuet-1-′TesA E.coli Accessions: ′TesA NP_415027, EC: 3.1.1.5, 3.1.2.— pETDuet-1-TEucUmbellularia californica Q41635 pBluescript-TEuc UcFatB1 pMAL-c2X-TEucAAA34215 pETDuet-1-TEch Cuphea hookeriana ABB71581 pBluescript-TEchChFatB2 AAC49269 pMAL-c2X-TEch ChFatB3 AAC72881 pETDuet-1-TEccCinnamonum camphorum pBluescript-TEcc CcFabB AAC49151 TEcipETDuet-1-atFatA3 Arabidopsis thaliana NP_189147 pETDuet-1-HaFatA1Helianthus annuus AAL769361 pCDFDuet-1-fadD-acr1 E. coli fadD:Accessions NP_416319, EC 6.2.1.3 acr1: Accessions YP_047869pETDuet-1-′TesA E. coli Accessions: ′TesA NP_415027, EC: 3.1.1.5,3.1.2.— pETDuet-1-TEuc Umbellularia californica Q41635 pBluescript-TEucUcFatB1 AAA34215 pMAL-c2X-TEuc pETDuet-1-TEch Cuphea hookeriana ABB71581pBluescript-TEch ChFatB2 AAC49269 pMAL-c2X-TEch ChFatB3 AAC72881pETDuet-1-TEcc Cinnamonum camphorum pBluescript-TEcc CcFatB AAC49151TEci pCDFDuet-1-fadD-acr1 E. coli fadD: Accessions NP_416319, EC 6.2.1.3acr1: Accessions YP_047869

One of ordinary skill in the art will appreciate that different plasmidsand genomic modifications can be used to achieve similar strains.

The selected expression plasmids contained compatible replicons andantibiotic resistance markers to produce a four-plasmid expressionsystem.

In some embodiments, LS9001 can be co-transformed with: (i) any of theTE-expressing plasmids; (ii) the fadD-expressing plasmid, which alsoexpresses acr1; and (iii) ester synthase expression plasmid.

As will be clear to one of ordinary skill in the art, when LS9001 isinduced with IPTG, the resulting strain will produce increasedconcentrations of fatty alcohols from carbon sources such as glucose.

Example 3. Production of Fatty Alcohol in the Recombinant E. coli Strain

Fatty alcohols were produced by expressing a thioesterase gene and anacyl-CoA reductase gene exogenously in a production host. Morespecifically, plasmids pCDFDuet-1-fadD-acr1 (acyl-CoA reductase) andpETDuet-1-'TesA (thioesterase) were transformed into E. coli strainLS9001 and corresponding transformants were selected using LB platessupplemented with 100 mg/L spectinomycin and 50 mg/L carbenicillin. Fourtransformants of LS9001/pCDFDuet-1-fadD-acrl were independentlyinoculated into 3 mL of an M9 medium supplemented with 50 mg/Lcarbenicillin and 100 mg/L spectinomycin. The samples containing thetransformants were cultured at 25° C. in a shaker (shaking at about 250rpm) until they reached 0.5 OD₆₀₀. Next, 1.5 mL of each sample wastransferred into a 250 mL flask containing 30 mL of the M9 mediumdescribed above. The resulting culture was grown at 25° C. in a shakeruntil it reached an OD₆₀₀ of between 0.5-1.0. IPTG was then added to afinal concentration of 1 mM. Cell growth continued for 40 hours.

The cells were then centrifuged and pelleted at 4,000 rpm. The cellpellet was suspended in 1.0 mL of methanol. 3 mL of ethyl acetate wasthen mixed with the suspended cells, followed by the addition of 3 mL ofH₂O. Next, the mixture was sonicated for 20 minutes. The resultingsample was centrifuged at 4,000 rpm for 5 minutes. Then the organicphase (the upper phase), which contained fatty alcohol(s), was subjectedto GC/MS analysis. The total alcohol (including tetradecanol,hexadecanol, hexadecenol, and octadecenol) titer was about 1-10 mg/L.When an E. coli strain carrying only empty vectors was cultured underthe same conditions and following the same protocol, a fatty alcoholstiter of only 0.2-0.5 mg/L was obtained.

Example 4. Production of Fatty Acids (FA) and Fatty Acid Ethyl Esters(FAEE) Containing Odd-Numbered Carbon Chains without Heavy Metals

1. Production of Biodiesel Sample #23-30

Biodiesel sample #23-30 (“sample #23-30”) was produced by bioreactorcultivation of an E. coli strain (C41 DE3 ΔfadE ΔfabR 'TesA fadD adp1ws)engineered to produce fatty esters. A two-stage inoculum protocol wasutilized for expansion of the culture. The first stage consisted of theinoculation of a 50 mL LB medium (supplemented with 100 μg/Lcarbenicillin and 100 μg/L spectinomycin) in a 250 mL baffled shakeflask with a 1 mL frozen stock vial of the E. coli ester productionstrain. This seed flask was incubated at 37° C. for about 7 hours (finalOD₆₀₀=4.5, pH 6.7), after which 3 mL of the primary culture wastransferred to each of three 2 L baffled flasks containing 350 mLbuffered F1 minimal medium that also contained 100 μg/L carbenicillinand 100 μg/L spectinomycin. The shake flask buffer used was Bis-Trispropane at a final concentration of 200 mM (pH 7.2). These secondaryseed flasks were incubated at 37° C. for about 18 hours (final OD₆₀₀=12,pH 5.5) and the contents were used to inoculate three 14 L bioreactorswith a starting volume of 6.5 liters of buffered F1 minimal mediumfollowing inoculation. These bioreactors also contained 100 μg/Lcarbenicillin and 100 g/L spectinomycin.

These 14 L bioreactors were initially cultivated at 37° C., and thedissolved oxygen levels were maintained at 30% of saturation, using theagitation and oxygen enrichment cascade loops. The pH of thefermentation mix was maintained at 7.2, using 1 M H₂SO₄ and anhydrousammonia gas. A nutrient feed consisting primarily of 43% (w/v) glucosewas initiated in each bioreactor when the original 5 g/L glucose chargein the basal medium was exhausted. The glucose solution feed rate wasthen manually adjusted for the duration of the fermentation run to keepthe residual glucose at a low (but non-zero) value for the duration ofthe fermentation run. Cultures were induced with a final concentrationof 1 mM IPTG when the OD₆₀₀ of the cultures reached 30. At thisinduction point, the bioreactor cultivation temperature was reduced to30° C., and about 15 mL/L (on a 6.5 to 7-Liter volume basis) of ethanolwas added to the culture and monitored by HPLC throughout. Additionalethanol was added periodically to the bioreactors to maintain theresidual concentrations at about 20 mL/L. The contents of thebioreactors were harvested after about 60 hours of cultivation, withabout 10 L of the broth harvested from each of the three bioreactors.

These harvest broths were combined and extracted with an equivalentvolume of ethyl acetate with stirring at room temperature for two hours.The broth extracts were then centrifuged (3,500 rpm, 30 minutes) toseparate the liquid layers, followed by the removal of the organic layerfor further processing. Ethyl acetate was almost completely removed(<0.3% residual, as determined by GC/FID) from the organic layer usingrotary evaporation (Büchi, R-200), leaving about 90 mL of a dark, oilyliquid. This liquid was referred to as sample #23-30.

2. Quantification of FA and FAEE in Sample #23-30

GC-MS was performed using an Agilent 5975B MSD system equipped with a 30m×0.25 mm (0.10 μm film) DB-5 column. The column temperature was3-minute isothermal at 100° C. The temperature of the column wasprogrammed to rise from 100° C. to 320° C. at a rate of 20° C./min. Whenthe final temperature of 320° C. was reached, the column remainedisothermal for 5 minutes at that temperature. The injection volume was 1μL. The carrier gas, helium, was released at 1.3 mL/min. The massspectrometer was equipped with an electron impact ionization source. Theionization source temperature was set at 300° C. FAEE standards (e.g.,ethyl dodecanoate, ethyl tetradecanoate, ethyl cis-9-hexadecenoate,ethyl hexadecanoate, ethyl octadecanoate, all >99%); fatty acid methylester (FAME) standards (e.g., methyl dodecanoate, methyl tetradecanoate,methyl pentadecanoate, methyl cis-9-hexadecenoate, methyl hexadecanoate,methyl cis-11-octadecenoate, all >99%); trimethylsilyl diazomethane(TMSD, 2 M in hexane); hydrochloric acid (37%); methanol (>99.9%); andethyl acetate (>99.9%) were purchased from Sigma-Aldrich and appliedwithout prior purification.

Sample #23-30 was derivatized by adding 50 μL trimethylsilyldiazomethane(TMSD), 8 μl HCl, and 36 μl methanol to 1 mL of sample (1 mg/mL in ethylacetate). The mixture was incubated at room temperature for 1 hour.

Prior to quantitation, the FAEE and FAME in sample #23-30 wereidentified using two methods. First, the GC retention time of eachcompound was compared to the retention time of a known standard. Second,identification of each compound was confirmed by matching the compound'smass spectrum to a standard's mass spectrum in the mass spectra library.

When a standard for a FAEE or FAME was available, the quantification ofthe FAEE or FAME was determined by generating a calibration curve(concentration vs. instrument response). A linear relationship betweenthe instrument response and the analyte concentration was then obtained.The concentration of the compound in the sample was determined by takingits instrument response and referring to the calibration curve.

When a standard for an FAEE was not available, an average instrumentresponse was used to determine the compound's concentrations. The slopeand the intercept for all existing calibration curves were averaged.From these averages, a linear relationship between concentration andinstrument response was determined. The concentrations of unknowncompounds were then determined by referencing the instrument responsesto the linear relationship between instrument response and concentrationusing Equation 1.

concentration=(instrument response−average interception)/averageslope  Equation 1:

After identifying and quantifying the FAME, the concentration of theassociated free fatty acids was determined based upon the concentrationof FAME and the molecular weight ratio of FA to FAME. Finally, theconcentration of FAEE and FA in mg/L was converted into percentage inthe biodiesel sample (w/w %).

The concentrations of FAEE and FA in sample #23-30 are listed in Table8. The total concentration of FAEEs and FAs was 80.7%. The rest of theunknown compounds may be analyzed by LC/MS/MS method. Ethylpentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate and ethylcis-11-octadecenoate were the major component of sample #23-30.

TABLE 8 Percentage of FAEE and FA in sample #23-30 Name Structure MWPercentage, % Ethyl dodecanoate

228.2 1.82 ± 0.03 Ethyl tridecanoate

242.2 0.16 ± 0.01 Ethyl tetradecanoate

256.2 12.88 ± 0.16  Ethyl pentadecanoate

270.3 0.62 ± 0.02 Ethyl cis-9-hexadecenoate

282.3 24.12 ± 0.20  Ethyl hexadecanoate

284.3 9.04 ± 0.11 Ethyl heptadecanoate

298.3 0.11 ± 0.01 Ethyl cis-11-octadecenoate

310.3 23.09 ± 0.33  Ethyl octadecanoate

312.3 0.19 ± 0.03 Dodecanoic acid

200.2 0.94 ± 0.02 Tetradecanoic acid

228.2 2.63 ± 0.03 Pentadecanoic acid

242.2 0.10 ± 0.01 cis-9-hexadecenoic acid

254.2 1.97 ± 0.01 Hexadecanoic acid

256.2 1.01 ± 0.01 cis-11-octadecenoic acid

282.3 2.00 ± 0.02 *Percentage is w/w %.

Surprisingly, sample #23-30 contained odd-numbered FA and FAEE.

3. Quantitative Elemental Analysis of Sample #23-30

Heavy metals are known to poison the catalysts used in catalyticcracking. To measure the levels of heavy metals in sample #23-30, sample#23-30 was sent to Galbraith Laboratories, Inc., for quantitativeelemental analysis of arsenic, calcium, carbon, chlorine, cobalt,copper, hydrogen, iron, Karl Fisher water, lead, manganese, magnesium,mercury, molybdenum, nitrogen, potassium, sodium, sulfur, zinc, oxygen,and phosphorus. Preparatory and analytical methods are described below.Results are shown in Table 9. All amounts in Table 9 were below thelevel of quantitation (LOQ) except for carbon (73.38%), chlorine (91ppm), hydrogen (12.1%), Karl Fisher water (0.998%), mercury (0.057 ppm),oxygen (14.53%), and phosphorus (343 ppm). Therefore, sample #23-30 didnot contain high levels of the heavy metals of concern.

Method G-52, Rev 6: Microwave Digestion of Samples for Metals Analysis

An appropriate amount of sample was weighed into a microwave vessel tothe nearest 0.001 g. The appropriate reagents were then added to themicrowave vessel. If a visible reaction was observed the reaction wasallowed to cease before capping the vessel. The vessel was then sealedand placed in the microwave according to the manufacturer's directions.The temperature of each vessel reached a minimum of 180±10° C. in 5minutes. It remained at a minimum of 180±10° C. for 10 minutes. At theend of the microwave program the vessels were allowed to cool for aminimum of 5 minutes before removal. The vessels were then uncapped andtransferred to volumetric flasks for analysis by the proper technique.

Method G-55, Rev 3: Parr Oxygen Bomb Combustion for the Determination ofHalogens

Samples were weighed into a combustion cup, and mineral oil was added asa combustion aid. For chlorine (Cl) and bromine (Br) measurements, 1%hydrogen peroxide solution was added into the bomb. For sulfur (S)measurements, a 0.01 N sodium hydroxide solution was added. The sampleand cup were sealed into a Parr oxygen combustion bomb along with asuitable absorbing solution. The bomb was purged with oxygen, thenpressurized to 25-30 atm of oxygen pressure, and ignited. Afterwards,the contents of the bomb were well mixed and transferred to a beaker forsubsequent analysis.

Method G-30B, Rev 7: Wet Ash Digestion of Inorganic and OrganicCompounds for Metals Analysis

The sample was charred using H₂SO₄. If analyzing for metals that forminsoluble sulfates, HClO₄ and HNO₃ were used to char the organicmaterial. After charring the sample, HNO₃ was added and the sample wasrefluxed to solubilize the metals present. If the solution becamecloudy, HCl was added to aid complete digestion. HF can be used ifsilicon was present in the sample but only if silicon was not an analyteof interest. All HF used was restricted to Teflon vessels. The cleardigestate was quantitatively transferred to a Class A volumetric flaskand brought to final volume. The sample was then analyzed.

Method ME-4A Rev 2: Determination of Anions Suppressed by IonChromatography

Instrument Dionex Model DX500 Chromatograph Column Dionex IonPac AS9-SC4 × 250 mm Eluent 2.4 mM Na₂CO₃ 1.8 mM NaHCO₃ Preparation Aqueoussamples may be analyzed as is. Water-soluble samples are typicallytransferred by weight to a known volume. Other solid materials that arenot water-soluble may be extracted to determine extractable quantitiesof various anions or combusted to determine total quantities of anelement such as Cl or Br. Calibration Standards to bracket sampleconcentration. 0.2 mg/L-4.0 mg/L Sample Intro Auto injection (HitachiModel AS7200) Determination Conductivity detection/linear regressionQuantitation Limit Typically 0.2 mg/L in solution. Interferences Anionswith similar retention times; overlapping peaks from major constituentanions.

Method S-300 Rev 7: Determination of Water by Coulometric Titration(Karl Fischer)

This method combined coulometry with the Karl Fischer titration. Thesample was mixed with an amine-methanol mixture containing predominantlyiodide ion (I−) and sulfur dioxide. The iodine produced at the anodethrough the electrolysis was allowed to react with water. In such cases,iodine was produced in direct proportion to the quantity of electricityaccording to Faraday's Law. Also, because 1 mole of waterstoichiometrically reacts with 1 mole of iodine, 1 mg of water wasequivalent to 10.71 coulombs of electricity. Utilizing this principle,the Moisture Meter determined the amount of water directly from thenumber of coulombs required for the electrolysis. This procedureincluded both direct introduction and a vaporizer pre-treatmenttechnique.

Preparation Weigh to obtain 100 μg to 3 mg H2O; Protect samples fromatmospheric moisture during weighing and transfer. Instrument MitsubishiMoisture Meter MCl Model CA-06 (Inst. #569) Mitsubishi MoistureVaporizer, Model CA/ VA-06 (Inst. #568) Control Sodium tartratemonohydrate (15.66%); Frequency: every 10 samples, one each day minimum,95-105% recovery Sample Intro A. Entry port, Direct transfer; capillary,syringe, or scoop B. Furnace, tin capsules (Water Vaporizer VA-06);Temperature varies, 200° C. is default value used for standards. Mostsamples analyzed at 160° C. Other temperatures upon request.Determination Coulometric titration of Karl Fischer reagent viaautomatic titrator Quantitation Limit 100 μg H₂O Precision & AccuracyRSD RE INSTR# Sodium Tartrate 1.35% −0.54% 569 Monohydrate 1.34% −2.13%568 Equations (2I⁻ − 2e⁻ → I₂); (I₂ + SO₂ + 3C₅H₅N + H₂O → 2C₅H₅N HI +C₅H₅N SO₃) μg H₂O/spl wt (g) = ppm H₂O μgH₂O × 0.1/spl wt (mg) = % H₂)Interferences (direct transfer only) free alkali; oxidizing, reducingagent; mercaptans

Method E16-2, Rev 9 (Trace E16-2A): Sulfur Determination Using the LECOSC-432DR

The SC-432DR Sulfur Analyzer is a non-dispersive infrared, digitallycontrolled instrument designed to measure sulfur content in a variety oforganic and inorganic materials. The sample was combusted at 1350±50° C.in an atmosphere of pure oxygen. The sulfur was oxidized to sulfurdioxide and quantitated by infrared absorption. The SC-432DR wasequipped with two detectors, a high-range and a low-range infrared cell.

Instrument LECO SC-432DR Sulfur Analyzer Sample Intro Weigh sample tonearest 0.01 mg. Weigh samples directly into sample boat tared onelectronic balance. Weight automatically transferred to SC432 database.Cover sample with LECO Com-Cat combustion accelerator as called for bysample type. Calibration Three conditioners of 5-10 mg cystine. Sevencalibration standards of 30-175 mg NIST SRM 8415 Whole Egg Powder(0.512% S). Internal calibration using a quadratic regressed curve.Control NIST SRM 1549 Milk Powder (0.351%); others to match sample type.Frequency: one for every ten samples. Determination Combustion in O₂atmosphere at 1350° C. Determination of resulting SO₂ by infra- reddetector. Quantitation 0.08 mg S Limit Calculations Internal Precision &Accuracy RSD (%) Mean Recovery (%) (milk powder) 2.60 97.97

Method ME-2, Rev 14: Carbon, Hydrogen, and Nitrogen Determination

This instrument burns sample in pure oxygen at 950° C. under staticconditions to produce combustion products of CO₂, H₂O, and N₂. ThePE-240 automatically analyzes these products in a self-integrating,steady state thermal conductivity analyzer. Tungstic anhydride may beadded to aid combustion. An extended combustion time (e.g., burn hardmode) may be employed for difficult to combust samples.

Instrument PerkinElmer 240 Elemental Analyzer (Instrument # 409, 410)Sample intro Weigh 1.0-2.5 mg into Al capsule; crimp (see GLI ProcedureG-6) for liquids; washed with solvent prior to weighing upon requestDecomposition Combustion at ≥950° C., reduction at ≥675° C. = CO₂, H₂O,N₂ Calibration Cyclohexanone-2,4-dinitropheylhydrazone (1-2.5 mg)Control s-1409, 2-1410: Cyclohexanone-2,4-dinitropheylhydrazone (51.79%C, 5.07% H, 20.14% N) Determination CO₂, H₂O, N₂ by thermal conductivityanalyzer Quantitation 0.5% C, 0.5% H, 0.5% N Instrument #409 Instrument#410 Precision & accuracy C H N C H N RSD % 0.28 1026 0.39 0.35 1.120.41 Mean recovery (%) 99.94 101.25 99.86 100.13 100.40 100.04Interferences Metals and some halogens cause incomplete combustion.Combustion aids and/or an extended combustion time can be used toalleviate this problem. Calculations Instrument calculates & prints w/wresults for % C, % H, and % N. For samples crimped in an aluminumcapsule, the % N is corrected with a factor; (μV/μg sample/K) × 100 = %Element, where K = calibration = μV/μg of C, or H, or N

Method ME-70, Rev 4: Inductively Coupled Plasma Atomic EmissionSpectrometry

This method describes multi-elemental determinations by ICP-AES usingsimultaneous optical systems and axial or radial viewing of the plasma.The instrument measures characteristic emission spectra by opticalspectrometry. Samples were nebulized and the resulting aerosol wastransported to the plasma torch. Element-specific emission spectra wereproduced by radio-frequency inductively coupled plasma. The spectra weredispersed by a grating spectrometer, and the intensities of the emissionlines were monitored by photosensitive devices. Background correctionwas required for trace element determination. Background was measuredadjacent to analyte lines on samples during analysis. The positionselected for the background-intensity measurement, on either or bothsides of the analytical line, was determined by the complexity of thespectrum adjacent to the analyte line. In one mode of analysis, theposition used should be as free as possible from spectral interferenceand should reflect the same change in background intensity as occurs atthe analyte wavelength measured. Background correction is not requiredin cases of line broadening where a background correction measurementwould actually degrade the analytical result.

Instrument ICP-OES Optima 5300, 3300DV and 4300DV, or equivalentDecomposition Prior to analysis, samples must be acidified or digestedusing appropriate Sample Preparation Methods. Calibration 0.01 ppm-60ppm plus matrix specific calibrations Sample Intro Peristaltic pump,cross flow nebulizer, gemcone nebulizer, scott ryton spray chamber andquartz cylonic spray chamber Determination Atomic emission by radiofrequency inductively coupled plasma of element-specific emissionspectra through a grating spectrometer monitored by photosensitivedevices. Quantitation Element and calibration specific ranging fromLimit 0.01-2 ppm Precision & ±10% RSD Accuracy Interferences Spectral,chemical, physical, memory Calculations wt % = (fc × v/10 × D)/spl ppm =(fc × v × D)/SPL Where fc = final concentration in μg/mL; v = samplevolume in mL; D = dilution factor; spl = sample mass in mg; SPL = samplemass in g

Method E80-2, Rev 4: Determination of Mercury (Automated Cold VaporTechnique)

This procedure is based on EPA SW846 Method 7471A. Cold Vapor AtomicAbsorption is based on the general theory of atomic absorption, whichholds that free atoms of the analyte absorb energy from a lamp sourcethat is proportional to the concentration of analyte. By using a lampcontaining the metal to be measured, the exact wavelength needed forabsorption was produced and interferences were greatly reduced. ColdVapor Atomic Absorption uses this principle, and the mercury atoms wereliberated by reducing mercury ions with Tin (II) Chloride (SnCl₂).Nitrogen gas carried the atoms through an optical cell, with the Hg lampon one end and the detector on the other end. Because the cold vapormethod was employed, instead of a flame method, undigested organiccompounds were an interference concern, because of their wide band ofabsorption wavelengths.

Instrument PerkinElmer FIMS 400 Automated Mercury Analyzer or equivalentDecomposition Variable, usually microwave digestion or permanganate hotwater bath digestion Calibration 0.1-5.0 μg/L Sample IntroductionAutosampler, peristaltic pump Determination Primary wavelength 253.7 nm,using a solid state detector Detection Limit Varies with preparationmethod and sample matrix Precision & Accuracy For microwave digestion:For MnO₄ ⁻digestion: RE −2.47% 4.90% RSD  7.48% 5.20% InterferencesUndigested organic compounds Calculations${{ppb}\mspace{14mu}{Hg}} = \frac{{µg}\text{/}L\mspace{14mu}{in}\mspace{14mu}{solution} \times {volume}\mspace{14mu}({mL}) \times {dilution}\mspace{14mu}{factor}}{{sample}\mspace{14mu}{weight}\mspace{14mu}(g)}$

TABLE 9 Quantitative elemental analysis of sample #23-30 AnalyticalElement Preparation Method Method Result Arsenic G-52 ME-70 <25 ppmCalcium G-30B ME-70 <119 ppm Carbon N/A ME-2 73.38% Chlorine G-55 ME-4A91 ppm Cobalt G-30B ME-70 <23 ppm Copper G-30B ME-70 <23 ppm HydrogenN/A ME-2 12.1%  Iron G-30B ME-70 <136 ppm Karl Fisher water N/A S-300 0.998% Lead G-52 ME-70 <25 ppm Manganese G-30B ME-70 <23 ppm MagnesiumG-30B ME-70 <23 ppm Mercury G-52 E80-2 0.057 ppm Molybdenum G-30B ME-70<23 ppm Nitrogen N/A ME-2 <0.5%  Potassium G-30B ME-70 <103 ppm SodiumG-30B ME-70 <140 ppm Sulfur N/A E16-2A  <0.140% Zinc G-30B ME-70 <23 ppmOxygen N/A Subtraction* 14.53% Phosphorus G-30B ME-70 343 ppm Resultspresented as “<” are below LOQ. *Oxygen content was determined bysubtracting the observed results for all other elements from 100%.

Example 5. Production and Release of Fatty Alcohol from Production Host

acr1 (encoding acyl-CoA reductase) was expressed in E. coli culturedwith glucose as the sole carbon and energy source. The E. coli producedsmall amounts of fatty alcohols such as dodecanol (C_(12:0)—OH),tetradecanol (C_(14:0)—OH), and hexadecanol (C_(16:0)—OH). In othersamples, FadD (acyl-CoA synthase) was expressed together with acrl in E.coli. A five-fold increase in fatty alcohol production was observed.

In other samples, acr1, fadD, and accABCD (acetyl-CoA carboxylase), in aplasmid carrying accABCD constructed as described in EXAMPLE 1, wereexpressed along with various individual thioesterases (TEs) in wild-typeE. coli C41 (DE3) and an E. coli C41 (DE3 ΔfadE, a strain lackingacyl-CoA dehydrogenase). This resulted in further increases in fattyalcohol production and modulation of the profiles of fatty alcohols (seeFIG. 6). For example, over-expression of E. coli ^('TesA)(pETDuet-1-'TesA) in this system achieved about a 60-fold increase inC_(12:0)—OH, C_(14:0)—OH and C_(16:0)—OH, with C_(14:0)—OH being themajor fatty alcohol. A very similar result was obtained when the ChFatB3enzyme (FatB3 from Cuphea hookeriana in pMAL-c2X-TEcu) was expressed.When the UcFatB1 enzyme (FatB1 from Umbellularia californicain inpMAL-c2X-TEuc) was expressed, fatty alcohol production increased about20-fold and C_(12:0)—OH was the predominant fatty alcohol.

Expression of ChFatB3 and UcFatB1 also led to the production ofsignificant amounts of the unsaturated fatty alcohols C_(16:1)—OH andC_(14:1)—OH, respectively. Fatty alcohols were also found in thesupernatant of samples generated from the expression of 'tesA. At 37°C., about equal amounts of fatty alcohols were found in the supernatantand in the cell pellet. Whereas at 25° C., about 25% of the fattyalcohols was found in the supernatant. See FIG. 7.

Example 6. Production of Fatty Alcohol Using a Variety of Acyl-CoAReductases

This example describes fatty alcohol production using a variety ofacyl-CoA reductases. Fatty alcohols can be the final product.Alternatively, the production host cells can be engineered toadditionally express/overexpress ester synthases to produce fattyesters.

Each of four genes encoding fatty acyl-CoA reductases (Table 10) fromvarious sources were codon-optimized for E. coli expression andsynthesized by Codon Devices, Inc. (Cambridge, Mass.). Each of thesynthesized genes was cloned as an NdeI-AvrII fragment intopCDFDuet-1-fadD vector (described in Example 2). Each of the plasmidscarrying these acyl-CoA reductase genes with the E. coli fadD gene wastransformed into E. coli strain C41 (DE) strain (purchased fromOver-expression).

The recombinant strains were cultured in 3 mL of an LB broth(supplemented with 100 mg/L spectinomycin) at 37° C. overnight. 0.3 mLof the overnight culture was transferred to 30 mL of a fresh M9 medium(containing 100 mg/L spectinomycin) and cultured at 25° C. When thecultures reached OD₆₀₀ of 0.5, 1 mM IPTG was added. Each culture was fed0.1% of one of three fatty acids dissolved in H₂O at pH 7.0. The threefatty acids fed were sodium dodecanoate, sodium myristate, or sodiumpalmitate. A culture without the addition of fatty acid was alsoincluded as a control. After induction, the cultures were allowed togrow at the same temperature for an additional 40 hours at 25° C.

The quantification of fatty alcohol yield at the end of fermentation wasperformed using GC-MS as described above in EXAMPLE 3 and/or EXAMPLE 4.The resulting fatty alcohol produced from the corresponding fatty acidis shown in Table 11. The results indicated that three acyl-CoAreductases—Acrl, AcrM, and BmFAR—were able to convert all three fattyacids into corresponding fatty alcohols. The results also indicated thathFAR and JjFAR had activity when myristate and palmitate were thesubstrates. However, there was little or no activity when dodecanoatewas the substrate. mFAR1 and mFAR2 only demonstrated low activity withmyristate and demonstrated no activity with the other two fatty acids.

TABLE 10 Acyl-CoA reductases Protein ID Acyl-CoA reductase Accessionnumber Protein sources mFAR1 AAH07178 Mus musculus mFAR2 AAH55759 Musmusculus JjFAR AAD38039 Simmondsia chinensis BmFAR BAC79425 Bombyx moriAcr1 AAC45217 Acinetobacter baylyi ADP1 AcrM BAB85476 Acinetobacter sp.M1 hFAR AAT42129 Homo sapiens

TABLE 11 Fatty alcohol production Peak Area^(c) Dodeca- No fattyAcyl-CoA noate/ Myristate/ Palmitate/ acid E. coli reductase dodeca-tetra- hexa- feeding ^(a)/ C41(DE3) genes nol^(b) decanol^(b)decanol^(b) hexadecanol mFAR1 7,400 85,700 8,465 70,900 mFAR2 2,90014,100 32,500 25,800 JjFAR 5,200 8,500 53,112 33,800 BmFAR 35,800409,000 407,000 48,770 acr1 202,000 495,000 1,123,700 58,515 acrM 42,500189,000 112,448 36,854 hFAR1 5,050 59,500 109,400 94,400 vector control4,000 1,483 32,700 27,500 media control 10,700 1,500 25,700 25,000 Note:^(a) Only hexadecanol was quantified in this case. ^(b)Fatty acidfed/fatty alcohol produced. ^(c)The area peak of fatty alcohol produced.

Example 7. Medium Chain Fatty Esters

Alcohol acetyl transferases (AATs, EC 2.3.1.84), which is responsiblefor acyl acetate production in various plants, can be used to producemedium chain length fatty esters, such as octyl octanoate, decyloctanoate, decyl decanoate, and the like. Fatty esters, synthesized frommedium chain alcohol (such as C₆ and C₈) and medium chain acyl-CoA orfatty acids (such as C₆ and C₈) have relatively low melting points. Forexample, hexyl hexanoate has a melting point of about −55° C. and octyloctanoate has a melting point of about −18° C. to about −17° C. The lowmelting points of these compounds make them suitable for use asbiofuels.

In this example, an SAAT gene encoding a thioesterase was co-expressedin a production host E. coli C41(DE3, ΔfadE) (as described inInternational Application No. PCT/US08/058788, the disclosures of whichis incorporated herein by reference) with fadD from E. coli and acr1(alcohol reductase from A. baylyi ADP1). Octanoic acid was provided inthe fermentation broth. This resulted in the production of octyloctanoate. Similarly, when the ester synthase gene from A. baylyi ADP1was expressed in the production host instead of the SAAT gene, octyloctanoate was produced.

A recombinant SAAT gene was synthesized by DNA 2.0 (Menlo Park, Calif.94025). The synthesized DNA sequence was based on the published genesequence (GenBank Accession No. AF193789), but modified to eliminate theNcoI site. The synthesized SAAT gene (as a BamHI-HindIII fragment) wascloned in pRSET B (Invitrogen, Carlsbad, Calif.), linearized with BamHIand HindIII. The resulting plasmid, pHZ1.63A was cotransformed into anE. coli production host with pAS004.114B, which carries a fadD gene fromE. coli and acr1 gene from A. baylyi ADP1. The transformants werecultured in 3 mL of an M9 medium containing 2% glucose. After IPTGinduction and the addition of 0.02% octanoic acid, the culture wasallowed to grow at 25° C. for 40 hours. 3 mL of acetyl acetate was thenadded to the whole culture and mixed several times using a mixer. Theacetyl acetate phase was analyzed by GC/MS.

Surprisingly, no acyl acetate was observed in the acetyl acetateextract. However, octyl octanoate was observed. However, the controlstrain without the SAAT gene (C41(DE3, ΔfadE)/pRSET B+pAS004.114B) didnot produce octyl octanoate. Furthermore, the strain (C41(DE3,ΔfadE)/pHZ1.43 B+pAS004.114B) in which the ester synthase gene from A.baylyi ADP1 was carried by pHZ1.43 produced octyl octanoate (see FIGS.8A-D).

The finding that SAAT activity produces octyl octanoate makes itpossible to produce medium chain fatty esters, such as octyl octanoateand octyl decanoate, which have low melting points and are suitable foruse as biofuels and for replacing triglyceride based biodiesel.

Example 8. Production of Fatty Esters in E. coli Strain Ls9001

Fatty esters were produced by engineering an E. coli production host toexpress a fatty alcohol forming acyl-CoA reductase, thioesterase, and anester synthase. Thus, the production host produced both the A and the Bside of the ester and the structure of both sides was influenced by theexpression of the thioesterase gene.

The LS9001 strain was transformed with plasmids carrying an estersynthase gene from A. baylyi ADP1 (plasmid pHZ1.43), a thioesterase genefrom Cuphea hookeriana (plasmid pMAL-c2X-Tech), and a fadD gene from E.coli (plasmid pCDFDuet-1-fad).

Plasmid pHZ1.43 carrying the ester synthase (WSadp1, GenBank AccessionNo. AA017391, EC 2.3.175) was constructed as follows. First the gene forWsadp1 was amplified with the following primers using genomic DNAsequence from A. baylyi ADP1 as template:

WSadp1_NdeI, (SEQ ID NO: 35) 5′-TCATATGCGCCCATTACATCCG-3′; andWSadp1_Avr, (SEQ ID NO: 36) 5′-TCCTAGGAGGGCTAATTTAGCCCTTTAGTT-3′.

Then, the PCR product was digested with NdeI and AvrII and cloned intopCOLADuet-1 to give pHZ 1.43. The plasmid carrying wSadpl was thenco-transformed into E. coli strain LS9001 with both pETDuet-1'TesA andpCDFDuet-1-fadD-acr/, and transformants were selected on LB platessupplemented with 50 mg/L of kanamycin, 50 mg/L of carbenicillin and 100mg/L of spectinomycin.

Three transformants were inoculated in 3 mL of LBKCS (LB brothsupplement with 50 mg/L kanamycin, 50 mg/L carbenicillin, 100 mg/Lspectinomycin, and 10 g/L glucose) and incubated at 37° C. in a shaker(shaking at 250 rpm). When the cultures reached an OD₆₀₀ of about 0.5,1.5 mL of each culture was transferred into 250 mL flasks containing 50mL LBKCS. The flasks were then incubated in a shaker (250 rpm) at 37° C.until the culture reached an OD₆₀₀ of about 0.5 to about 1.0. IPTG wasthen added to a final concentration of 1 mM. The induced cultures wereincubated at 37° C. in a shaker (250 rpm) for another 40-48 hours.

The cultures were then transferred into 50 mL conical tubes and thecells were centrifuged at 3,500×g for about 10 minutes. Each of the cellpellets was then mixed with 5 mL ethyl acetate. The ethyl acetateextracts were analyzed with GC/MS. The titer of fatty esters (includingC₁₆C₁₆, C_(14:1)C₁₆, C_(18:1)C_(18:1), C₂C₁₄, C₂C₁₆, C₂C_(16:1),C16C_(16:1) and C₂C₁₈:i) was about 10 mg/L. When an E. coli strain onlycarrying empty vectors was cultured under the same conditions andfollowing the same protocol, only 0.2 mg/L fatty esters was found in theethyl acetate extract.

Example 9. Production and Release of Fatty-Ethyl Ester from ProductionHost

The LS9001 strain was transformed with plasmids carrying an estersynthase gene from A. baylyi (plasmid pHZ1.43), a thioesterase gene fromCuphea hookeriana (plasmid pMAL-c2X-TEcu) and a fadD gene from E. coli(plasmid pCDFDuet-1-fadD).

This recombinant strain was cultured at 25° C. in 3 mL of an M9 mediumcontaining 50 mg/L kanamycin, 100 mg/L carbenicillin, and 100 mg/Lspectinomycin. After IPTG induction, the medium was adjusted to a finalconcentration of 1% ethanol and 2% glucose.

The culture was allowed to grow for 40 hours after IPTG induction. Thecells were separated from the spent medium by centrifugation at 3,500×gfor 10 minutes. The cell pellet was re-suspended with 3 mL of the M9medium. The cell suspension and the spent medium were then extractedwith 1 volume of ethyl acetate. The resulting ethyl acetate phases fromthe cell suspension and the supernatant were subjected to GC-MSanalysis.

The C₁₆ ethyl ester was the most prominent ester species for thisthioesterase and 20% of the fatty ester produced was released from thecell. See FIG. 9. A control E. coli strain C41(DE3, ΔfadE) containingpCOLADuet-1 (empty vector for the ester synthase gene), pMAL-c2X-TEuc(containing fatB from U. california) and pCDFDuet-1-fadD (fadD gene fromE. coli) failed to produce detectable amounts of fatty acid ethylesters. The fatty acid esters were quantified using commercial palmiticacid ethyl ester as the reference.

Fatty esters were also made using the methods described herein exceptthat methanol or isopropanol was added to the fermentation broth. Theexpected fatty esters were produced.

Example 8. The Influence of Various Thioesterases on the Composition ofFatty-Ethyl Esters Produced in Recombinant E. coli Strains

The thioesterases FatB3 (C. hookeriana), 'TesA (E. coli), and FatB (U.california) were expressed simultaneously with ester synthase (from A.baylyi). A plasmid, pHZ1.61, which comprises a pCDFDuet-1 (Novagen,Madison, Wis.) backbone with the fadD gene, was constructed by replacingthe NotI-AvrII fragment (carrying the acr1 gene) with the NotI-AvrIIfragment from pHZ1.43 such that fadD and the ADP1 ester synthase were inone plasmid and each of the coding sequences was under the control of aseparate T7 promoter. The construction of pHZ1.61 made it possible touse a two-plasmid system instead of the three-plasmid system. pHZ1.61was then co-transformed into E. coli C41(DE3, ΔfadE) with one of theplasmids, each carrying a different thioesterase gene as describedherein.

The total fatty acid ethyl esters (in both the supernatant andintracellular fatty acid ethyl fluid) produced by these transformantswere evaluated using the technique described herein. The titers and thecomposition of fatty acid ethyl esters are summarized in Table 12.

TABLE 12 Titers (mg/L) and composition of fatty acid ethyl esters byrecombinant E. coli C41(DE3, ΔfadE)/pHZ1.61 and plasmids carryingvarious thioesterase genes. Thioesterases C₂C₁₀ C₂C_(12:1) C₂C₁₂C₂C_(14:1) C₂C₁₄ C₂C_(16:1) C₂C₁₆ C₂C_(18:1) Total ‘TesA 0.0 0.0 6.5 0.017.5 6.9 21.6 18.1 70.5 ChFatB3 0.0 0.0 0.0 0.0 10.8 12.5 11.7 13.8 48.8ucFatB 6.4 8.5 25.3 14.7 0.0 4.5 3.7 6.7 69.8 pMAL 0.0 0.0 0.0 0.0 5.60.0 12.8 7.6 26.0 Note: ‘TesA, pETDuet-1-’TesA; chFatB3, pMAL-c2X-TEcu;ucFatB, pMAL-c2X-TEuc; pMAL, pMAL-c2X, the empty vector for thioesterasegenes used in the study.

Example 9. Use of Various Ester Synthases to Produce Biofuel

Four genes encoding ester synthases were synthesized based oncorresponding polynucleotide sequences reported in NCBI GenBank withminor modifications. These modifications include the removal of internalNcoI, NdeI, HindIII, and AvrII restriction sites without introducingother changes to the corresponding amino acid sequence. The four genesof interest were each synthesized with an NdeI site on the 5′ end and anAvrII at the 3′ end. The sequences were then cloned into the NdeI andAvrII site of pCOLADuet-1 (Novagene) to produce pHZ1.97-376,pHZ1.97-377, pHZ1.97-atfA1 and pHZ1.97-atfA2. The plasmids carrying eachof the four genes of interest along with the respective GenBankAccession numbers and the GenPeptide Accessions numbers are listed inTable 13 below.

TABLE 13 Ester synthases GenBank GenPeptide DNA sequence Accessionaccession Plasmids ID original sources No. No. pHZ1.97- FES376Marinobacter CP000514.1 ABM17275 376 (376) aquaeolei VT8 pHZ1.97- FES377Marinobacter CP000514.1 ABM20141 377 (377) aquaeolei VT8 pHZ1.97- FESA1Alcanivorax NC_008260.1 YP_694462 atfA1 (AtfA1) borkumensis SK2 pHZ1.97-FESA2 Alcanivorax NC_008260.1 YP_693524 atfA2 (AtfA2) borkumensis SK2

Each of the four plasmids was transformed into E. coli C41 (DE3,ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadD. Three transformants fromeach transformation were selected for fermentation studies to determinetheir abilities to synthesize fatty acid ethyl esters. The fermentationstep was performed as described in EXAMPLE 6, but at two differenttemperatures, 25° C. or 37° C. Strain C41 (DE3,ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadD+pHZ1.43 (expressing ADP1ester synthase) was used as a positive control and C41 (DE3,ΔfadEΔfabR)/pETDuet-1-'TesA+pCDFDuet-1-fadD as a negative control.

The expression of each of the four ester synthase genes in the E. colistrain with attenuated fadE and fabR activity and overexpressing 'tesAand fadD enabled each strain to produce about 250 mg/L of FAEE at 25° C.This was the same amount produced by the positive control that expressedADP1 ester synthase. In contrast, the negative control strain producedless than 50 mg/L FAEE under the same conditions at 25° C. (see, FIG.10). The fatty acyl composition of FAEE produced from these four estersynthases was similar to that from ADP1 ester synthases (see, FIG. 11)

Results from fermentations performed at 37° C. indicated that strainscarrying pHZ1.97_aftA2 and strains carrying pHZ1.97_376 produced moreFAEE than the positive control carrying pHZ1.43 (see, FIG. 12). Thestrains carrying pHZ1.97_aftA2 and the strains carrying pHZ1.97_376 alsoproduced large amount of free fatty acid (see, FIG. 13). Whereas thestrain carrying pHZ.143 did not accumulate free fatty acid. The resultsdemonstrated that these four ester synthases were capable of acceptingethanol and a broad range of acyl-CoA as substrates.

Example 12. Use of Eukaryotic Ester Synthase to Produce Biofuel

This example describes the cloning and expression of an ester synthasefrom Saccharomyces cerevisiae. Plasmids were generated using standardmolecular biology techniques.

TABLE 14 Plasmids with eeb1 Vector Given Name Backbone ConstructionpGL10.59 pCOLADuet-1 eeb1* gene inserted between BamHI (Novagen) andHindIII sites (KanR) pGL10.104 pMAL c2x eeb1* gene inserted betweenBamHI (NEB) and HindIII sites (AmpR) pMAL-c2X-TEuc pMAL c2x See Table 7above (NEB) pCDFDuet-1-acr1 pCDFDuet-1 See Table 7 above (Novagen) *TheSaccharomyces cerevisiae gene eeb1 (GenBank Accession number YPL095C)was PCR-amplifed from S. cerevisiae genomic DNA sequence using primersthat introduced the 5′ BamHI and 3′ HindIII sites.

An E. coli C41 (DE3 ΔfadE) production host was used to express thevarious plasmids. The E. coli cells were cultured in an M9 minimalmedium (containing 6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/LNH₄Cl, 1 mg/L thiamine (vit. B1), 1 mM MgSO₄, 0.1 mM CaCl₂, 0.4% (w/v)or 2% (w/v) glucose). All fatty acid stock solutions were prepared bydissolving the fatty acid sodium or potassium salt in distilleddeinoized water at pH 7.0. Octanoic acid stock was purchased from Sigma,St. Louis, Mo.

Fermentations were performed using the C41 (DE3 ΔfadE) strain containingplasmids pCDFDuet-1-acr1, pMAL-c2X-TEuc (ucFatB), and pGL10.59 (eebl).The control strain was C41 (DE3 ΔfadE) strain carrying pCDFDuet-1-acr1,pMAL-c2X-TEuc, and the empty pCOLADuet-1 vector. Each of the threecolonies from each transformation were used to inoculate an M9+0.4%glucose starter culture supplemented with carbenicillin (100 μg/mL),spectinomycin (100 μg/mL), and kanamycin (50 μg/mL). The cultures wereallowed to grow at 37° C. overnight. Production cultures wereestablished by making a 1:100 dilution of starter culture to inoculate 3mL M9 media+0.4% glucose. The production cultures were allowed to growat 37° C. until OD₆₀₀=0.6 before being induced with 1 mM IPTG, fed 1%ethanol, and cultured for an additional 40 hours at 25° C. Whole cellcultures were extracted with an equal volume of ethyl acetate byvortexing vigorously for 30 seconds. The organic phase was taken andexamined on the GC/MS using the method alkane_1_splitless_ctc.m for FAEEdetection, which is described above in EXAMPLE 4, part 2,“Quantification of FA and FAEE in sample #23-30.”

No FAEE peaks were detected in the samples. In order to determinewhether eebl was correctly expressed, IPTG-induced and uninducedcultures were analyzed by SDS-PAGE. No band corresponding to the size ofEeb1 (about 52 kDa) was detected. This suggested that, for thisparticular plasmid system, Eeb1 was not well-expressed.

Additional expression experiments were performed using a differentexpression vector. The gene was cloned into the vector pMALc2x, whichexpressed the target protein as a maltose binding protein (MBP) fusion.SDS-PAGE analysis of whole-cell lysates revealed that cultures inducedwith 1 mM IPTG yielded an appropriately-sized band corresponding to theEeb1-MBP fusion (about 92 kDa). The band was not present in uninducedcells. This experiment was described in detail in InternationalApplication No. PCT/U.S. Ser. No. 08/058,788, the disclosures therein isincorporated by reference in the entirety.

Eeb1 enzymatic activity was assessed using the C41 (DE3 ΔfadE) E. colistrain carrying plasmids pCDFDuet-1-acr1 and pGL10.104 (eeb1). A C41(DE3 ΔfadE) with pCDFDuet-1-acr1 and pMALc2x served as the controlstrain. Three colonies were picked from each transformation and each wasused to inoculate an M9+0.4% glucose overnight starter culturesupplemented with carbenicillin (100 μg/mL) and spectinomycin (100μg/mL). A 1:100 dilution of the starter culture was used to inoculate 10mL of an M9+0.4% glucose production cultures. The production cultureswere allowed to grow at 37° C. until OD₆₀₀=0.4-0.5 before inducing with1 mM IPTG. The cultures were each fed about 1% ethanol, octanoic acid(to about 0.01% or about 0.02% of the final volume), and/or decanoicacid (to about 0.02% of the final volume). Fermentations were allowed tocontinue for 24 hours at 25° C. Extractions were carried out by adding1/10 volume of 12 M HCl and an equal volume of ethyl acetate to theculture and vortexing for 30 seconds. Samples were analyzed by GC/MS asdescribed above.

GC/MS data revealed a peak corresponding to the octanoic acid ethylester can be detected for cells expressing eeb1 and fed octanoic acidand ethanol. The vector control strain also showed a C₂C₈ peak, albeit asmaller peak than that of the eeb1-expressing cells.

Cells that were fed 0.02% decanoic acid did not grow well; therefore,the following studies were conducted using 0.01% or 0.005% decanoicacid. To test the ability of Eebl to utilize alcohols other than ethanolin synthesizing fatty acid esters, fermentations were carried out usingthe same strain: C41 (DE3 ΔfadE) with pCDFDuet-1-acr1 and pGL10.104.Cells were cultured as previously described. At induction, the cellswere fed 0.02% octanoic acid along with 1% methanol, ethanol, propanol,or isopropanol. Cells were also fed 0.01% or 0.005% decanoic acid and 1%ethanol. Fermentations were allowed to continue post-induction for 24hours at 25° C. To prepare for analysis by GC/MS, cultures werecentrifuged to separate the pellet and the supernatant. The pellet wasresuspended in an equal volume of a fresh M9+0.4% glucose medium. Boththe resuspended pellets and supernatant samples were extracted asdescribed above and analyzed by GC/MS.

All of the supernatant samples contained large amounts of fatty acid butno detectable fatty acid esters. Similarly, the vector control pelletsamples contained no fatty acid ester peaks, as determined using GC/MS.However, cells fed a C₁₀ fatty acid showed peaks that were identified asrepresenting decanoic acid.

The pellet samples derived from the cells expressing Eeb1 and fed a C₈fatty acid and propanol or ethanol showed small peaks corresponding topropyl or ethyl esters. No peak was detected from the cells that werefed methanol or isopropanol. Cultures fed 0.01% or 0.005% of a C₁₀ fattyacid and ethanol also produced a C₂C₁₀ FAEE, but the FAEE was found inthe pellet samples.

The results indicated that Eeb1 was capable of synthesizing FAEEs usingoctanoic or decanoic acids, and was also able to use methanol togenerate the octanoic methyl ester. However, these compounds were highlyvolatile and as such the GC/MS data might not have accurately reflectedthe true titers. To more accurately measure product formation ahexadecane overlay was used to facilitate the capture of more volatileFAEEs.

Eeb1 activity with regard to fatty acid substrates was assessed usingstrain C41 (DE3 ΔfadE) with pCDFDuet-1-acr1 and pGL10.104, which was feddifferent chain-length fatty acids. Cells were cultured as describedabove, but were induced at OD₆₀₀=0.8-0.9 so as to promote better cellgrowth post-induction. At this point, cells were fed 1% ethanol and0.02% of a C₈ fatty acid or 0.01% of a combination of the followingfatty acids: C₁₀, C₁₂, C₁₄, and C₁₆. Cultures that were fed C₈ or C₁₀fatty acids were overlaid with 20% total volume of hexadecane.Fermentations were carried out for an additional 24 hours at 25° C. postinduction. For product analysis, whole cultures (without separating thesupernatant from the pellet) were extracted as described herein, with1/10 volume of HCl and an equal volume (to the volume of the culture) ofethyl acetate. Hexadecane-treated samples were injected directly intothe GC/MS using the program hex_1_splitless_ctc.m, which is describedabove in EXAMPLE 4, part 2, “Quantification of FA and FAEE in sample#23-30.”

None of the vector controls had any detectable FAEE peaks. For the C₈-and C₁₀-fed cells, large C₂C₈ and C₂C₁₀ peaks were detected in thehexadecane samples, but not in the ethyl acetate samples. Thisdemonstrated that hexadecane was able to successfully trap the volatileFAEEs. For the rest of the ethyl acetate samples, small peaks weredetected for C₂C₁₂ and C₂C₁₄ FAEEs, but no C₂C₁₆ FAEE was detected.Thus, Eeb1 generated ethyl esters using fatty acids with chain lengthsfrom C₈ to C₁₄. Eeb1 favored C₈ and C₁₀ over the longer-chain fattyacids.

Example 13. Genomic Integration of Recombinant Sequences to Make a HostStrain that Over-Expresses E. coli FabA and/or FabB Genes

It is known that the product of the fabR gene acts as a repressor of theexpression of the fabA and fabB genes. It is also known that FadR worksas an activator of the same genes. The FabR and predicted consensusbinding sequences were previously published by Zhang et al., J. Biol.Chem. 277: 15558-15565, 2002. The consensus binding sequences and theirlocations relative to the fabA and fabB genes of E. coli is shown inFIG. 14.

A fabR knockout strain of E. coli was created. Primers TrmA_R_NotI andFabR_FOP were used to amplify about 1,000 bp upstream of fabR, andprimers SthA_F_Bam and FabR_ROP were used to amplify about 1000 bpdownstream of fabR. Overlap PCR was applied to create a construct forin-frame deletion of the complete fabR gene. The fabR deletion constructwas cloned into a temperature-sensitive plasmid pKOV3, which containedSacB for counterselection. A chromosomal deletion of fabR was madeaccording to the method described in Link et al., J. Bact.,179:6228-6237, 1997.

TABLE 15 fabR knock-out primers Primer Name Primer Sequence (5′ to 3′)TrmA_R_Not ATAGTTTAGCGGCCGCAAATCGAGCTGGATCAGGAT TA (SEQ ID NO: 37)FabR_FOP AGGATTCAGACATCGTGATGTAATGAAACAAGCAAATC AAGATAGA (SEQ ID NO: 38) SthA_F_Bam CGCGGATCCGAATCACTACGCCACTGTTCC(SEQ ID NO: 39) FabR_ROP TTGATTTGCTTGTTTCATTACATCACGATGTCTGAATCCTTG (SEQ ID NO: 40)

Example 14. Production Host Construction

Table 16 identifies the homologs of certain genes described herein,which are known to be expressed in microorganisms that producebiodiesels, fatty alcohols, and hydrocarbons. To increase fatty acidproduction and, therefore, hydrocarbon production in production hostssuch as those identified in Table 16, heterologous genes can beexpressed, such as those from E. coli.

One of ordinary skill in the art will appreciate that genes that areendogenous to the microorganisms provided in Table 16 can also beexpressed, over-expressed, or attenuated using the methods describedherein. Moreover, genes that are described in Table 16 can be expressed,overexpressed, or attenuated in production hosts that endogenouslyproduce hydrocarbons to allow for the production of specifichydrocarbons with defined carbon chain length, saturation points, andbranch points.

TABLE 16 Hydrocarbon production hosts Organism Gene Name AccessionNo./SEQ ID/Loci EC No. Desulfovibrio desulfuricans accA YP_3880346.4.1.2 G20 Desulfovibrio desulfuricans accC YP_388573/YP_3880336.3.4.14, 6.4.1.2 G22 Desulfovibrio desulfuricans accD YP_388034 6.4.1.2G23 Desulfovibrio desulfuricans fabH YP_388920 2.3.1.180 G28Desulfovibrio desulfuricans fabD YP_388786 2.3.1.39 G29 Desulfovibriodesulfuricans fabG YP_388921 1.1.1.100 G30 Desulfovibrio desulfuricansacpP YP_388922/YP_389150 3.1.26.3, G31 1.6.5.3, 1.6.99.3 Desulfovibriodesulfuricans fabF YP_388923 2.3.1.179 G32 Desulfovibrio desulfuricansgpsA YP_389667 1.1.1.94 G33 Desulfovibrio desulfuricans ldhAYP_388173/YP_390177 1.1.1.27, G34 1.1.1.28 Erwinia (micrococcus) accA942060-943016 6.4.1.2 amylovora Erwinia (micrococcus) accB3440869-3441336 6.4.1.2 amylovora Erwinia (micrococcus) accC3441351-3442697 6.3.4.14, 6.4.1.2 amylovora Erwinia(micrococcus) accD2517571-2516696 6.4.1.2 amylovora Erwinia (micrococcus) fadE1003232-1000791 1.3.99.— amylovora Erwinia (micrococcus) plsB(D311E)333843-331423 2.3.1.15 amylovora Erwinia (micrococcus) aceE840558-843218 1.2.4.1 amylovora Erwinia (micrococcus) aceF 843248-8448282.3.1.12 amylovora Erwinia (micrococcus) fabH 1579839-1580789 2.3.1.180amylovora Erwinia (micrococcus) fabD 1580826-1581749 2.3.1.39 amylovoraErwinia (micrococcus) fabG CAA74944 1.1.1.100 amylovora Erwinia(micrococcus) acpP 1582658-1582891 3.1.26.3, amylovora 1.6.5.3, 1.6.99.3Erwinia (micrococcus) fabF 1582983-1584221 2.3.1.179 amylovora Erwinia(micrococcus) gpsA 124800-125810 1.1.1.94 amylovora Erwinia(micrococcus) ldhA 1956806-1957789 1.1.1.27, amylovora 1.1.1.28Kineococcus radiotolerans accA ZP_00618306 6.4.1.2 SRS30216 Kineococcusradiotolerans accB ZP_00618387 6.4.1.2 SRS30216 Kineococcusradiotolerans accC ZP_00618040/ 6.3.4.14, 6.4.1.2 SRS30216 ZP_00618387Kineococcus radiotolerans accD ZP_00618306 6.4.1.2 SRS30216 Kineococcusradiotolerans fadE ZP_00617773 1.3.99.— SRS30216 Kineococcusradiotolerans plsB(D311E) ZP_00617279 2.3.1.15 SRS30216 Kineococcusradiotolerans aceE ZP_00617600 1.2.4.1 SRS30216 Kineococcusradiotolerans aceF ZP_00619307 2.3.1.12 SRS30216 Kineococcusradiotolerans fabH ZP_00618003 2.3.1.180 SRS30216 Kineococcusradiotolerans fabD ZP_00617602 2.3.1.39 SRS30216 Kineococcusradiotolerans fabG ZP_00615651 1.1.1.100 SRS30216 Kineococcusradiotolerans acpP ZP_00617604 3.1.26.3, SRS30216 1.6.5.3, 1.6.99.3Kineococcus radiotolerans fabF ZP_00617605 2.3.1.179 SRS30216Kineococcus radiotolerans gpsA ZP_00618825 1.1.1.94 SRS30216 Kineococcusradiotolerans ldhA ZP_00618879 1.1.1.28 SRS30216 Rhodospirillum rubrumaccA YP_425310 6.4.1.2 Rhodospirillum rubrum accB YP_427521 6.4.1.2Rhodospirillum rubrum accC YP_427522/YP_425144/ 6.3.4.14, 6.4.1.2YP_427028/YP_426209/ YP_427404 Rhodospirillum rubrum accD YP_4285116.4.1.2 Rhodospirillum rubrum fadE YP_427035 1.3.99.— Rhodospirillumrubrum aceE YP_427492 1.2.4.1 Rhodospirillum rubrum aceF YP_4269662.3.1.12 Rhodospirillum rubrum fabH YP_426754 2.3.1.180 Rhodospirillumrubrum fabD YP_425507 2.3.1.39 Rhodospirillum rubrum fabGYP_425508/YP_425365 1.1.1.100 Rhodospirillum rubrum acpP YP_4255093.1.26.3, 1.6.5.3, 1.6.99.3 Rhodospirillum rubrum fabFYP_425510/YP_425510/ 2.3.1.179 YP_425285 Rhodospirillum rubrum gpsAYP_428652 1.1.1.94 1.1.1.27 Rhodospirillum rubrum ldhAYP_426902/YP_428871 1.1.1.28 Vibrio furnissii accA  1, 16 6.4.1.2 Vibriofurnissii accB  2, 17 6.4.1.2 Vibrio furnissii accC  3, 18 6.3.4.14,6.4.1.2 Vibrio furnissii accD  4, 19 6.4.1.2 Vibrio furnissii fadE  5,20 1.3.99.— Vibrio furnissii plsB(D311E)  6, 21 2.3.1.15 Vibriofurnissii aceE  7, 22 1.2.4.1 Vibrio furnissii aceF  8, 23 2.3.1.12Vibrio furnissii fabH  9, 24 2.3.1.180 Vibrio furnissii fabD 10, 252.3.1.39 Vibrio furnissii fabG 11 ,26 1.1.1.100 Vibrio furnissii acpP12, 27 3.1.26.3, 1.6.5.3, 1.6.99.3 Vibrio furnissii fabF 13, 282.3.1.179 Vibrio furnissii gpsA 14, 29 1.1.1.94 Vibrio furnissii ldhA15, 30 1.1.1.27, 1.1.1.28 Stenotrophomonas maltophilia accA ZP_016437996.4.1.2 R551-3 Stenotrophomonas maltophilia accB ZP_01644036 6.4.1.2R551-3 Stenotrophomonas maltophilia accC ZP_01644037 6.3.4.14, 6.4.1.2R551-3 Stenotrophomonas maltophilia accD ZP_01644801 6.4.1.2 R551-3Stenotrophomonas maltophilia fadE ZP_01645823 1.3.99.— R551-3Stenotrophomonas maltophilia plsB(D311E) ZP_01644152 2.3.1.15 R551-3Stenotrophomonas maltophilia aceE ZP_01644724 1.2.4.1 R551-3Stenotrophomonas maltophilia aceF ZP_01645795 2.3.1.12 R551-3Stenotrophomonas maltophilia fabH ZP_01643247 2.3.1.180 R551-3Stenotrophomonas maltophilia fabD ZP_01643535 2.3.1.39 R551-3Stenotrophomonas maltophilia fabG ZP_01643062 1.1.1.100 R551-3Stenotrophomonas maltophilia acpP ZP_01643063 3.1.26.3 R551-3 1.6.5.3,1.6.99.3 Stenotrophomonas maltophilia fabF ZP_01643064 2.3.1.179 R551-3Stenotrophomonas maltophilia gpsA ZP_01643216 1.1.1.94 R551-3Stenotrophomonas maltophilia ldhA ZP_01645395 1.1.1.28 R551-3Synechocystis sp. PCC6803 accA NP_442942 6.4.1.2 Synechocystis sp.PCC6803 accB NP_442182 6.4.1.2 Synechocystis sp. PCC6803 accC NP_4422286.3.4.14, 6.4.1.2 Synechocystis sp. PCC6803 accD NP_442022 6.4.1.2Synechocystis sp. PCC6803 fabD NP_440589 2.3.1.39 Synechocystis sp.PCC6803 fabH NP_441338 2.3.1.180 Synechocystis sp. PCC6803 fabFNP_440631 2.3.1.179 Synechocystis sp. PCC6803 fabG NP_440934 1.1.1.100,3.1.26.3 Synechocystis sp. PCC6803 fabZ NP_441227 4.2.1.60 Synechocystissp. PCC6803 fabl NP_440356 1.3.1.9 Synechocystis sp. PCC6803 acpNP_440632 Synechocystis sp. PCC6803 fadD NP_440344 6.2.1.3 Synechococcuselongates accA YP_400612 6.4.1.2 PCC7942 Synechococcus elongates accBYP_401581 6.4.1.2 PCC7942 Synechococcus elongates accC YP_4003966.3.4.14, PCC7942 6.4.1.2 Synechococcus elongates accD YP_400973 6.4.1.2PCC7942 Synechococcus elongates fabD YP_400473 2.3.1.39 PCC7942Synechococcus elongates fabH YP_400472 2.3.1.180 PCC7942 Synechococcuselongates fabF YP_399556 2.3.1.179 PCC7942 Synechococcus elongates fabGYP_399703 1.1.1.100, PCC7942 3.1.26.3 Synechococcus elongates fabZYP_399947 4.2.1.60 PCC7942 Synechococcus elongates fabl YP_3991451.3.1.9 PCC7942 Synechococcus elongates acp YP_399555 PCC7942Synechococcus elongates fadD YP_399935 6.2.1.3 PCC7942

The Accession Numbers of Table 16 are from GenBank, Release 159.0 as ofApr. 15, 2007, EC Numbers of Table 16 are from KEGG, Release 42.0 as ofApril 2007 (plus daily updates up to and including May 9, 2007), resultsfor Erwinia amylovora strain Ea273 were obtained from the Sangersequencing center, completed shotgun sequence as of May 9, 2007,positions for Erwinia represent locations on the Sangerpsuedo-chromosome, sequences from Vibrio fumisii M1 are from the VFM1pseudo-chromosome, v2 build, as of Sep. 28, 2006, and include the entiregene, and may also include flanking sequence.

Example 15. Additional Exemplary Production Strains

Table 17 provides additional exemplary production strains. Two examplebiosynthetic pathways are described for producing fatty acids, fattyalcohols, and wax esters. For example, Table 17 provides examples 1 and2 that produce fatty acids. The production host strain used to producefatty acids in example 1 is a production host cell that is engineered tohave the desired synthetic enzymatic activities. Each “x” marks thegenes correlated to the activities, for example, acetyl-CoA carboxylase,thio-esterase, and acyl-CoA synthase activity. Production host cells canbe selected from bacteria, yeast, and fungi. As provided in Table 17,additional production hosts can be created using the indicated exogenousgenes.

TABLE 17 Combination of genes useful for making genetically engineeredproduction strains II. Fatty acids III. Fatty alcohols IV. wax/fattyesters Peptide Sources of genes Genes example 1 example 2 example 1example 2 example 1 example 2 acetyl-CoA E. coli accABCD X X X X X Xcarboxylase thio- E. coli ‘TesA X X X X esterase Cinnamomum camphoraccFatB Umbellularia californica umFatB X X Cuphea hookeriana chFatB2Cuphea hookeriana chFatB3 Cuphea hookerian chFatA Arabidopsis thalianaAtFatA1 Arabidopsis thaliana AtFatB1 [M141T] acyl-CoA E. coli fadD X X XX X X synthase acyl-CoA Bombyx mori bFAR reductase Acinetobacter baylyiADP1 acr1 X X Simmondsia chinesis jjFAR X X Triticum aestivum TTA1 Musmusculus mFAR1 Mus musculus mFAR2 Acinetobacter sp M1 acrM1 Homo sapienshFAR Ester synthase/ Fundibacter jadensis WST9 alcohol DSM 12178 acyl-Acinetobacter sp. HO1-N WSHN X transferase Acinetobacter baylyl WSadp1 XADP1 Mus musculus mWS Homo sapiens hWS Fragaria × ananassa SAAT Malus ×domestica MpAAT Simmondsia chinensis JjWS (AAD38041) DecarbonylaseArabidopsis thaliana cer1 Oryzasativa cer1 Transport Acinetobacter sp.HO1-N unknown X X protein Arabidopsis thaliana Cer5

Example 16. Use of Additional Acyl-CoA Synthases to Over ProduceAcyl-CoA

Homologs to E. coli fadD can be expressed in E. coli by synthesizingcodon-optimized genes based on a desired sequence from M. tuberculosisHR7Rv (NP_217021, FadDD35), B. subtilis (NP_388908, YhfL), Saccharomycescerevisiae (NP_012257, Faa3p) or P. aeruginosa PAO1 (NP_251989). Thesynthetic genes can be designed to include NcoI- and HindII-compatibleoverhangs. The acyl-CoA synthases can then be cloned into a NcoI/HindIIIdigested pTrcHis2 vector (Invitrogen Corp., Carlsbad, Calif.) asdescribed above and expressed in E. coli strain MG1655 ΔfadE. Theexpression in E. coli may lead to an increased production of acyl-CoA.

Fatty acid derivatives such as an FAEE can also be produced byco-transformation of the E. coli strain MG1655 ΔfadE with variousacyl-CoA synthases in the pTrcHis2 vector with a compatible plasmidderived from pCL1920, which contains the thioester gene from Cupheahookeriana with or without an ester synthase from A. baylyi. Theresulting production host will produce FAEE when cultured in a mediumcontaining ethanol as described above.

Example 17. Use of Additional Acyl-CoA Synthases to Overproduce Acyl-CoA

DNA sequences or protein sequences of many E. coli FadD homologs areknown. However the biochemical properties of only a few have beendescribed. See, e.g., Knoll et al., J. Biol. Chem. 269(23):16348-56,1994; Shockey et al., Plant Physiol. 132: 1065-1076, 2003. Furthermore,their capacity to be expressed in an active form at sufficiently highlevels for commercial purposes is unknown. To explore the possibility ofusing heterologous acyl-CoA synthases for esters production, severalacyl-CoA synthase genes were cloned and expressed as follows. Althoughthis example describes transforming the production host with separateplasmids for the thioesterase, ester synthase, and acyl-CoA synthasegenes, these genes may alternatively be incorporated in a single plasmidto transform the production host.

1. Construction of pOP-80 Plasmid

To over-express the genes, a low-copy plasmid based on the commercialvector pCL1920 (Lerner and Inouye, NAR 18: 4631, 1990) carrying a strongtranscriptional promoter was constructed by digesting pCL1920 withrestriction enzymes AflII and SfoI (New England BioLabs Inc. Ipswich,Mass.). Three DNA sequence fragments were produced by this digestion.The 3737 bp fragment was gel-purified using a gel-purification kit(Qiagen, Inc. Valencia, Calif.). In parallel, a fragment containing thetrc-promoter and lacI region from the commercial plasmid pTrcHis2(Invitrogen, Carlsbad, Calif.) was amplified by PCR using primers LF302:5′-ATATGACGTCGGCATCCGCTTACAGACA-3′(SEQ ID NO:41); and LF303:5′-AATTCTTAAGTCAGGAGAGCGTTCACCGACAA-3′(SEQ ID NO:42). These two primersalso introduced recognition sites for the restriction enzymes ZraI(gacgtc) and AflII(cttaag), respectively, at the end of the PCRproducts. After amplification, the PCR products were purified using aPCR-purification kit (Qiagen, Inc. Valencia, Calif.) and digested withZraI and AflII following the conditions recommended by the manufacturer(New England BioLabs Inc., Ipswich, Mass.). After digestion, the PCRproduct was gel-purified and ligated with the 3737 bp DNA sequencefragment derived from pCL1920.

After transformation with the ligation mixture in TOP10 chemicallycompetent cells (Invitrogen, Carlsbad, Calif.), transformants wereselected on Luria agar plates containing 100 μg/mL spectinomycin. Manycolonies were visible after overnight incubation at 37° C. Plasmidspresent in these colonies were purified, analyzed with restrictionenzymes, and then sequenced. One plasmid produced in this way wasretained, named pOP-80, and used for further experiments. A map ofpOP-80 is shown in FIG. 16.

The DNA sequences of relevant regions of plasmid pOP-80 were verified.It was found in the junctions where the 2 fragments were ligated that 3to 4 bases at each end were missing. This was probably caused by anexonuclease activity contaminating one of the restriction enzymes. Itwas likely that these small deletions did not affect any relevantplasmid function. The resulting plasmid was used for all expressionexperiments described in this example. The full sequence of the plasmidis disclosed as SEQ ID NO:1 in FIG. 17.

2. Cloning of fadD35 from Mycobacterium tuberculosis HR7Rv

An E. coli codon-optimized gene was synthesized by DNA 2.0 Inc. (MenloPark, Calif.), using the protein sequence of the fadD35 gene depositedat NCBI with the GenBank Accession No. NP_217021 as a starting point.The synthetic gene contained a unique NcoI site at the 5′-end and aunique EcoRI site at the 3′-end. The synthetic gene was provided by DNA2.0 Inc. cloned in plasmid pJ201:16084. The fad35 gene was released fromthis plasmid by digesting with NcoI and EcoRI. The sequence of thisfragment is shown in SEQ ID NO:2 in FIG. 18. The resulting DNA sequencefragment is disclosed in SEQ ID NO:2 was ligated with pOP-80, which waspreviously digested with NcoI and EcoRI. The ligation mixture wastransformed into TOP10 chemically competent cells (Invitrogen, Carlsbad,Calif.), which were then plated on Luria agar plates containing 100μg/mL spectinomycin and incubated at 37° C. overnight. Colonies thatappeared the next day were screened, and a strain containing the correctplasmid was identified. The plasmid was named pDS9.

3. Cloning of fadD1 from Pseudomonas aeruginosa PAO1

An E. coli codon-optimized gene was synthesized by DNA 2.0 Inc. (MenloPark, Calif.) using the protein sequence of the fadD1 gene deposited atNCBI with the GenBank Accession No. NP_251989 as a starting point. Thesynthetic gene contained a unique BspHI site at the 5′-end and a uniqueEcoRI site at the 3′-end. The synthetic gene was provided by DNA 2.0,Inc. and cloned in plasmid pJ201:16083. The fadD1 gene was released fromthis plasmid by digesting with BspHI and EcoRI. The sequence of thisfragment is shown in SEQ ID NO:3 in FIG. 19. The resulting DNA sequencefragment of SEQ ID NO:3 was ligated with pOP-80, which was previouslydigested with NcoI and EcoRI. The ligation mixture was transformed intoTOP10 chemically competent cells (Invitrogen, Carlsbad, Calif.), whichwere then plated on Luria agar plates containing 100 μg/mL spectinomycinand incubated at 37° C. overnight. Colonies that appeared the next daywere screened. A strain containing the correct plasmid was identified.The plasmid was named pDS8.

4. Cloning of yhfL from Bacillus subtilis

The yhfL gene was amplified by PCR using Bacillus subtilis 1168chromosomal DNA sequence as a template, and two primers designed basedon the DNA sequence deposited at NCBI with GenBank Accession No.NC_000964. The sequences of the 2 primers were:

BsyhfLBspHIF: (SEQ ID NO: 4) 5′-CATCATGAATCTTGTTTC-3′ (FIG. 20)BsyhfLEcoR: (SEQ ID NO: 5) 5′- CGGAATTCTTATTGGGGCAAAATATC-3′ (FIG. 21)

These two primers introduced a BspHI recognition site at the 5′-end andan EcoRI recognition site at the 3′-end. The PCR product was cloneddirectly into pCR-Blunt II-TOPO vector using the Zero Blunt TOPO PCRcloning kit (Invitrogen, Carlsbad, Calif.). A plasmid carrying the yhfLgene was named pDS1. To subclone yhfL, plasmid pDS1 was digested withBspHI and EcoRI. The resulting DNA sequence fragment SEQ ID NO:6 (FIG.22) was gel-purified and cloned into pOP-80, which was previouslydigested with NcoI and EcoRI. The plasmid carrying the B. subtilis yhfLgene cloned into pOP-80 was named pDS4.

5. Cloning of faa3p from Saccharomyces cerevisiae (NP_012257)

The faa3p gene was amplified by PCR using commercial Saccharomycescerevisiae chromosomal DNA sequence ATCC 204508D (American Type CultureCollection, Manassas, Va.) as a template, and two primers that weredesigned based on the DNA sequence deposited at NCBI with the GenBankAccession No. NC_001141 as a template. The sequences of the two primerswere:

Scfaa3pPciF: (SEQ ID NO: 7) 5′-CGACATGTCCGAACAACAC-3′ (FIG. 23)Scfaa3pPciI: (SEQ ID NO: 8) 5′-GCAAGCTTCTAAGAATTTTCTTTG-3′ (FIG. 24)

These two primers introduced a PciI recognition site at the 5′-end and aHindIII recognition site at the 3′-end.

The PCR product was cloned directly into pCR-Blunt II-TOPO vector usingthe Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, Calif.). Aplasmid carrying the faa3p gene was named pDS2. To subclone faa3p,plasmid pDS2 was digested with PciI and HindIII. The DNA sequencefragment (SEQ ID NO:9) (FIG. 25) was gel-purified and cloned intopOP-80, which was previously digested with NcoI and HindIII. The plasmidcarrying the S. cerevisiae faa3p gene cloned into pOP-80 was named pDS5.

6. Cloning of ZP 01644857 from Stenotrophomonas maltophilia R551-3

The structural gene sequence for the protein ZP_01644857 is available atNCBI as part of the locus NZ_AAVZ01000044. The gene was amplified by PCRusing Stenotrophomonas maltophilia R551-3 chromosomal DNA sequence astemplate, and two primers designed based on the deposited DNA sequence.The sequences of the two primers were:

Smprk59BspF: (SEQ ID NO: 10) 5′- AGTCATGAGTCTGGATCG-3′ (FIG. 26)Smprk59HindR: (SEQ ID NO: 11) 5′- GGAAGCTTACGGGGCGGGCG-3′ (FIG. 27)

These two primers introduced a BspHI recognition site at the 5′-end anda HindIII recognition site at the 3′-end.

The PCR product was cloned directly into pCR-Blunt II-TOPO vector usingthe Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, Calif.). Aplasmid carrying the gene encoding the protein ZP_01644857 was namedpDS3. To facilitate further subcloning of the gene, an internal BspHIsite was removed by site directed mutagenesis using the primerPrkBsp:5′-GCGAACGGCCTGGTCTTTATGAAGTTCGGTGG-3′(SEQ ID NO:12) (FIG. 28)and the QuikChange Multi Site-Directed mutagenesis kit (Stratagene, LaJolla, Calif.). After the proper mutation was corroborated by DNAsequencing, the resulting plasmid was digested with BspHI and HindIII,and was named pDS6. The DNA sequence fragment was gel-purified andcloned into pOP-80 previously digested with NcoI and HindIII. Theplasmid carrying the gene encoding the protein ZP_01644857 cloned intopOP-80 was named pDS7. The protein sequence of ZP_01644857 is disclosedin FIG. 29 (SEQ ID NO:13).

7. Construction of Strains to Produce Fatty Esters.

An E. coli BL21(DE3) strain was first transformed with plasmidpETDuet-1-'TesA (described in EXAMPLE 2) carrying the E. coli 'tesAgene, and plasmid pHZ1.97 (described in EXAMPLE 9) carrying the atfA2ester synthetase gene, respectively. Both genes were under the controlof a T7 promoter inducible by IPTG. Two independent transformantscarrying both plasmids were transformed with each of the recombinantplasmids carrying the heterologous fadD genes, and selected on Luriaagar plates containing 100 μg/mL carbenicillin, 50 μg/mL kanamycin, and100 μg/mL spectinomycin. Three independent colonies carrying the threeplasmids were evaluated for fatty-ester production.

8. Analysis of Fatty Esters Produced Using ZP_01644857 fromStenotrophomonas maltophilia R551-3

To evaluate the use of the protein ZP_01644857 from Stenotrophomonasmaltophilia R551-3 in a production host to produce fatty esters, an E.coli BL21(DE3) strain was transformed with plasmid pETDuet-1-'TesA(described in EXAMPLE 2) carrying the E. coli 'tesA gene, plasmidpHZ1.97 (described in EXAMPLE 9) carrying the atfA2 ester synthetasegene, and plasmid pDS7 carrying the gene encoding the proteinZP_01644857 (described above in the instant example). This productionhost was fermented to produce fatty esters as described in EXAMPLE 4. Asa control, a second E. coli strain BL21(DE3)ΔfadE containing plasmidspETDuet-1-'TesA, pHZ1.97, and pCL1920 was used as a production host toproduce fatty esters.

Table 18 below indicates the fatty ester yields from these productionhosts.

TABLE 18 Fatty ester yields from a production host that producedZP_01644857 C₂C_(12:1) C₂C_(12:0) C₂C_(14:1) C₂C_(14:0) C₂C_(16:1)C₂C_(16:0) C₂C_(18:1) C₂C_(18:0) Total Ester type: mg/L mg/L mg/L mg/Lmg/L mg/L mg/L mg/L mg/L^(c) Control^(a) 0.0 0.0 0.0 1.78 9.80 5.65 33.70.00 50.93 fadD 1.49 3.57 3.68 33.22 52.77 43.09 91.11 10.08 239.01ZP_01644857 ^(b) ^(a)Control: strain BL21(DE3) D fadE, containingplasmids pETDuet-1-’TesA, pHZ1.97 and pCL1920. ^(b) Strain BL21(DE3) DfadE, containing plasmids pETDuet-1-’TesA, pHZ1.97 and pDS7. ^(c)Thesevalues represent the average of 3 cultures.

Example 18. Down-Regulation of Beta-Oxidation

This example describes the creation of an E. coli strain MG1655 ΔfadEΔydiO.

Fatty acid degradation can be eliminated or attenuated by attenuatingany of the β-oxidation enzymatic reactions described herein (see, FIG.2). For example, the E. coli strain MG1655 ΔfadE can be furtherengineered using primers to amplify up-stream of ydiO and additionalprimers to amplify downstream of ydiO. Overlap PCR can then be used tocreate a construct for in-frame deletion of the complete ydiO gene. TheydiO deletion construct is then cloned into a temperature sensitiveplasmid pKOV3, which contains a sacB gene for counter-selection. Achromosomal deletion of ydiO is then made according to the method ofLink et al., J. B act. 179:6228-6237, 1997. The resulting strain willnot be capable of degrading fatty acids and fatty acyl-COAs. Additionalmethods of generating a double knockout of fadE and ydiO are described,for example, in Campbell et al., Mol. Microbiol. 47:793-805, 2003.

It is also possible to avoid fatty acid degradation by selecting oremploying a production host that does not contain the I3-oxidationpathway. For example, several species of Streptococcus have beensequenced and no I3-oxidation genes have been found.

Example 19. Identification of Additional Ester Synthases

This example provides additional ester synthases and methods of usingsuch synthases for the production of fatty esters.

Using bioinformatics, additional ester synthases were identified. Theseester synthases contain motifs that differ from other known motifs, suchas the motifs found in ADP1. The differences in the motifs are noted inTable 19, below.

TABLE 19 Comparison of ester synthases motifs ADP1-motifs HHAXVDG NDVVLGALRXY PLXAMV ISNVPGP V A L P A REPLYXNG Hypothetical HHSLIDGY NDVALGGLRRF SLIVVLP VSNVPG EDVLYLRG protein A L P S BCG_3544c [Mycobacteriumbovis BCG str. Pasteur 1173P2] gi/121639399 Protein of HHALVDG NDVALGGLRKF SLIAFLP VSNVPG REPLYFNGS unknown Y A L P function UPF0089[Mycobacterium gilvum PYR-GCK] gi/145221651 Protein of HHALVDG NDVALGGLRKF SLIAFLP VSNVPG REPLYFNGS unknown Y A L P function UPF0089[Mycobacterium vanbaalenii PYR-1] gi/120406715

The identified sequences can be cloned using standard molecular biologytechniques. These sequences can be expressed using the vectors describedherein and used to make various fatty esters. The motifs can also beused to identify other ester synthases.

Example 20. Product Characterization

To characterize and quantify the fatty alcohols and fatty esters, gaschromatography (GC) coupled with electron impact mass spectra (MS)detection was used. Fatty alcohol samples were first derivatized with anexcess of N-trimethylsilyl (TMS) imidazole to increase detectionsensitivity. Fatty esters did not require derivatization. Fattyalcohol-TMS derivatives and fatty esters were dissolved in anappropriate volatile solvent, such as, for example, ethyl acetate.

The samples were analyzed on a 30 m DP-5 capillary column using thefollowing method. After a 1 μl splitless injection onto the GC/MScolumn, the oven was held at 100° C. for 3 minutes. The temperature wasincrementally raised to 320° C. at a rate of 20° C./minute. The oven washeld at 320° C. for an additional 5 minutes. The flow rate of thecarrier gas helium was 1.3 mL/minute. The MS quadrapole scanned from 50to 550 m/z. Retention times and fragmentation patterns of product peakswere compared with authentic references to confirm peak identity.

For example, hexadeconic acid ethyl ester eluted at 10.18 minutes (FIGS.15A-B). The parent ion of 284 mass units was readily observed. Moreabundant were the daughter ions produced during mass fragmentation. Themost prevalent daughter ion was of 80 mass units. The derivatized fattyalcohol hexadecanol-TMS eluted at 10.29 minutes and the parent ion of313 were observed. The most prevalent ion was the M-14 ion of 299 massunits.

Quantification was carried out by injecting various concentrations ofthe appropriate authentic references using the GC/MS method as describedherein. This information was used to generate a standard curve withresponse (total integrated ion count) versus concentration.

Example 21. Identification and Reclassification of a MicroorganismBelonging to the Genus Jeotgalicoccus that is an α-Olefin Producer

Micrococcus candicans ATCC 8456 was previously reported to synthesizealiphatic hydrocarbons with carbon chain lengths ranging from C₁₈ to C₂₀(Morrison et al., J. Bacteriol. 108:353-358, 1971). To identify thehydrocarbons produced by this strain, ATCC 8456 cells were cultured in15 mL TSBYE medium (3% Tryptic Soy Broth+0.5% Yeast Extract), for 40-48hours at 30° C. Cells from 5 mL of culture were pelleted, resuspended in1 mL methanol, sonicated for 30 minutes, and extracted with 4 mL hexane.After solvent evaporation, samples were resuspended in 0.1 mL hexane andanalyzed by GC-MS. The hydrocarbons were identified to be the followingα-olefins: 15-methyl-1-heptadecene (a-C₁₈), 16-methyl-1-heptadecene(i-C₁₈), 1-nonadecene (n-C₁₉), 17-methyl-1-nonadecene (a-C₂₉) and18-methyl-1-nonadecene (i-C₂₀) (see FIG. 34 (i=iso, a=anteiso,n=straight chain) and FIG. 36).

Based upon the following analyses, it was determined that ATCC 8456 waspreviously misidentified as belonging to the genus Micrococci. Thephylogenetic classification of ATCC 8456 was reassessed by amplifyingand sequencing the partial 16s rRNA gene using primers Eubac27 and 1492R(see DeLong et al., PNAS 89:5685, 1992). The 16s rRNA sequence ofATCC8456 was analyzed using the classifier program of the RibosomalDatabase Project II (http://rdp.cme.msu.edu/index.jsp). Based upon thisanalysis, the strain was identified as belonging to the genusJeotgalicoccus. The genus Jeotgalicoccus has been previously described(Jung-Hoon et al., Int. J. Syst. Evol. Microbiol. 53:595-602, 2003).

Additional analysis using the G+C content of ATCC 8456 was conducted.Jeotgalicoccus is a low G+C Gram-positive bacteria related to the genusStaphylococcus (see FIG. 37). Micrococci, on the other hand, are highG+C Gram-positive bacteria. The ends of several clones from a cosmidlibrary of ATCC 8456 genomic DNA were sequenced. Based upon a DNAsequence of about 4,000 bp, the G+C content was determined to be about36%. Nucleotide sequence searches against a non-redundant proteindatabase revealed that all sequences with a match to a database entrywere similar to proteins from low G+C Gram-positive bacteria, such asspecies belonging to the genus Staphylococcus or Bacillus, but notspecies belonging to the genus Micrococcus.

Next, an analysis of the entire genome of ATCC 8456 was conducted. Basedon a DNA sequence of about 2.1 MB, the G+C content of the entire genomewas determined to be about 36.7%. In contrast, bacteria of the genusMicrococcus are known to have high G+C genomes, e.g., the genome ofMicrococcus luteus NCTC 2665 has a G+C content of 72.9% (GenBankAccession No. ABLQ01000001-68). Based upon the G+C content analysis, itwas determined that the ATCC 8456 microorganism does not belong to thegenus Micrococcus.

Additional Jeotgalicoccus strains were also examined to determine ifthey produced α-olefins. The following strains of Jeotgalicoccus wereexamined: Jeotgalicoccus halotolerans DSMZ 17274, Jeotgalicoccuspsychrophilus DSMZ 19085, and Jeotgalicoccus pinnipedalis DSMZ 17030.Each strain was cultured in 15 mL TSBYE medium (3% Tryptic SoyBroth+0.5% Yeast Extract) and the hydrocarbons were isolated andanalyzed by GC-MS as described above. All three strains producedα-olefins similar to the ones produced by ATCC 8456 (FIGS. 34B, 34C and34D depict GC-MS traces for hydrocarbons produced by Jeotgalicoccushalotolerans DSMZ 17274 cells, Jeotgalicoccus pinnipedalis DSMZ 17030cells, and Jeotgalicoccus psychrophilus DSMZ 19085 cells, respectively).These data indicate that the ability to produce α-olefins is widespreadamong the genus Jeotgalicoccus.

Example 22. Production of Increased Levels of Olefins and α-Olefins notNormally Produced by ATCC 8456 Cells Using Fatty Acid Feeding

The fatty acids eicosanoic acid (straight-chain C₂₀ fatty acid),16-methyl octadecanoic acid and 17-methyl octadecanoic acid(branched-chain C₁₉ fatty acids) were identified as components of ATCC8456's lipids. These fatty acids were deduced to be the directprecursors, after decarboxylation, for 1-nonadecene,15-methyl-1-heptadecene and 16-methyl-1-heptadecene biosynthesis,respectively. In order to improve α-olefin production and to produceolefins not normally produced by ATCC 8456 cells, fatty acid feedingexperiments were carried out as described below.

ATCC 8456 cells were cultured in 15 mL of a TSBYE medium (containing 3%Tryptic Soy Broth+0.5% Yeast Extract). Fatty acids were added to theculture medium at a final concentration of 0.5 g/L (0.05%). After growthfor 40-48 hrs at 30° C., cells from 5 mL of culture were pelleted,resuspended in 1 mL methanol, sonicated for 30 minutes and extractedwith 4 mL hexane. After solvent evaporation, samples were resuspended in0.1 mL hexane and analyzed by GC-MS.

When cultures were fed eicosanoic acid, an increase in 1-nonadeceneproduction of about 18-fold was observed (see FIG. 38A; black tracesdepict without and gray traces depict with fatty acid feeding). Whencultures were fed stearic acid or palmitic acid, an increase in theproduction of the α-olefins 1-pentadecene and 1-heptadecene,respectively, was observed (see FIG. 38B). These olefins are notnormally produced by ATCC 8456 cells. This indicated that fatty acidswere the direct precursors for α-olefins and that Jeotgalicoccusbacteria can be used to enzymatically convert fatty acids into α-olefinsin vivo.

Alternatively, resting Jeotgalicoccus cells can be fed with variousfatty acids to achieve similar results.

Example 23. In Vitro Synthesis of α-Olefins Using Cell Extracts andPartially Purified Proteins

A cell free extract of ATCC 8456 was used to convert free fatty acidsinto α-olefins. The cell free extract was generated using the followingprocedure: ATCC 8456 cells were cultured in a TSBYE medium (containing3% Tryptic Soy Broth+0.5% Yeast Extract) at 30° C. for 24 hrs withshaking. The cells were then pelleted from the culture by centrifugationat 3,700 rpm for 20 minutes. The cell pellet was then resuspended in 50mM Tris buffer pH 7.5 with 0.1 M NaCl and 2.0 mM dithiothreitol to aconcentration of 0.1 g/mL cells. To this cell slurry, 200 units/mL oflysostaphin (Sigma) was added on ice. The cell lysis reaction continuedfor 30 minutes. The cells were then sonicated at 12 Won ice for threecycles of 1.5 seconds of sonication followed by 1.5 seconds of rest.Sonication lasted for a total of 9 seconds. This procedure was repeated5 times with a 1-minute interval between sonication cycles. The lysedcells were then subjected to centrifugation at 12,000 rpm for 10 minutesto pellet the cell debris. The supernatant (cell free extract) wasremoved and used for the conversion of free fatty acids to α-olefins.

After obtaining the cell free extract, the free fatty acids stearic acidand eicosanoic acid were converted to α-olefins using the cell freeextract as described below. First, a 5% stock solution of sodium orpotassium stearate was made in 1% Tergitol solution (Sigma, St. Louis,Mo.). Next, 6 μl of the stock solution was added to 1 mL of the cellfree extract at room temperature to obtain a final concentration of 1 mMfree fatty acid salt. The reaction was conducted at room temperature for3 hrs. The α-olefins were recovered by adding 200 μl of ethyl acetate tothe mixture, vortexing briefly, centrifuging briefly, and then removingthe organic phase. The α-olefins were identified and/or detected byGC/MS.

FIG. 39 shows the GC/MS trace for the resulting products. In sample 1,no stearic acid was added to the cell free extract. In sample 2, thecell free extract was replaced with 50 mM Tris pH 7.5 buffer with 0.1 Msodium chloride to which stearic acid was added. In sample, stearic acidwas added to the cell free extract. The peak at 7.62 minute had the sameretention time and the same mass spectra as 1-heptadecene (Sigma). Wheneicosanoic acid was added under similar conditions, 1-nonadecene wasformed.

Boiling the cell free extract eliminated the production of α-olefinsupon the addition of free fatty acids. This data strongly suggested thatthe ATCC 8456 catalyst was protein based.

The ATCC 8456 cell free extract did not require additional co-factors toproduce α-olefins. When the cell free extract was supplemented withseveral co-factors in 1 mM concentrations, no increase in α-olefinsynthesis was observed. The co-factors examined were NAD+, NADP+, NADH,NADPH, FADH₂, SAM, ATP, and CoA. In addition, Mg²⁺ was examined, but ata 10 mM concentration. The co-factor requirement was also tested bydialyzing the cell free extract with a 10 kDa cut-off membrane for 1.5hrs in a volume that was 200-fold greater than the cell extract volumeusing a dialysis buffer: 50 mM Tris, pH 7.5 with 0.1 M sodium chloride.No decrease in α-olefin synthesis was observed after dialysis.Additionally, no decrease in α-olefin synthesis was observed when 10 mMEDTA pH 7.5 was added to the reaction mixture.

The ATCC 8456 cell free extract was further enriched by carrying out anammonium sulfate precipitation. First, enough ammonium sulfate was addedto the cell free extract to bring the concentration of ammonium sulfateto 50% (wt/vol) saturation. The mixture was stirred gently on ice for 60minutes and then centrifuged at 13,000 rpm for 30 minutes. Thesupernatant was recovered and additional ammonium sulfate was added tobring the ammonium sulfate concentration to 65% (wt/vol). The mixturewas allowed to mix on ice for 60 minutes and was centrifuged again for30 minutes. The supernatant was discarded. The pellet was thenresuspended in 50 mM Tris buffer pH 7.5 with 0.1 M sodium chloride. Thismixture was then dialyzed in the aforementioned buffer to remove theammonium sulfate. The cell free extract treated with ammonium sulfatehad the same α-olefin synthesizing activity as the cell free extract.

Example 24. Purification and Identification of a Protein that ConvertsFatty Acids into α-Olefins

To isolate the protein necessary for α-olefin production from ATCC 8456cells, the following protein purification procedure was carried out.First, 6 L of ATCC 8456 cells were cultured in a TSBYE medium at 30° C.for 24 hours with shaking. The cells were pelleted by centrifugation at3,700 rpm for 20 minutes at 4° C., and the supernatant was discarded.The cell pellet was resuspended in a solution of 100 mL of 50 mM Tris pH8.0, 0.1 M NaCl, 2.0 mM DTT, and bacterial protease inhibitors. The cellslurry was then passed through a French press one time at a pressure of30,000 psi. Next, the cell slurry was sonicated as described in Example3 to shear the DNA. The cell free extract was centrifuged at 10,000 rpmfor 60 minutes at 4° C. The supernatant was then removed and ammoniumsulfate was added to a final concentration of 50% (wt/vol). The mixturewas gently stirred at 4° C. for 60 minutes and then centrifuged at10,000 rpm for 30 minutes. The supernatant was then removed andadditional ammonium sulfate was added to 65% (wt/vol) saturation. Themixture was stirred again for 60 minutes at 4° C. and centrifuged at10,000 rpm for 30 min. The supernatant was discarded. The remainingpellet was resuspended in 50 mL of 50 mM Tris pH 8.0 and 2.0 mM DTT.

The mixture was passed through a 5 mL HiTrap SP column (GE Healthcare)at 3 mL/min and 4° C. The following buffers were used as an elutiongradient: buffer A contained 50 mM Tris pH 8.0 and 2.0 mM DTT; buffer Bcontained 50 mM Tris pH 8.0, 1.0 M NaCl, and 2.0 mM DTT. After thecolumn was loaded with the mixture, the column was washed with 40%buffer B. Next a 20-minute gradient of 40% buffer B to 100% buffer B at3.0 mL/min was carried out. 5 mL fractions were collected during theelution gradient. Each fraction was tested for activity as described inExample 3. Fractions containing α-olefin production activity typicallyeluted between 600 and 750 mM NaCl concentration. Fractions containingactivity were then pooled and dialyzed into buffer A.

The dialyzed protein fraction was then loaded onto a 1 mL ResourceQ (GEHealthcare) column at 4 mL/min at 4° C. Buffer B used with the HiTrap SPcolumn was also used for the ResourceQ column. A 7-minute elutiongradient between 0% buffer B and 25% buffer B was run at 4 mL/min. 1.5mL fractions were collected and assayed for activity. Active fractionseluted between 150 and 200 mM NaCl concentrations. Fractions containingactivity were then pooled and concentrated with a Millipore Amiconprotein concentrator (4 mL and 10 kDa exclusion size) to about 50 μL.The approximate protein concentration was determined with a Bradfordassay (Bio-Rad). Final protein concentrations ranged from about 5 mg/mLto about 10 mg/mL. 30 μL of protein was then loaded onto a SDS PAGE gel(Invitrogen) along with an appropriate protein molecular weight marker.The gel was stained with Simple Safe Coomassie stain (Invitrogen). FIG.40 depicts a representative gel. Two intense protein bands at 50 kDa and20 kDa were observed.

To determine the identity of the protein bands, the bands were excisedfrom the gel, digested with trypsin, and analyzed using LC/MS/MS. TheLC/MS/MS data was analyzed using the program Mascot (Mann et al., Anal.Chem. 66:4390-4399, 1994). The ATCC 8456 genome was sequenced. Thegenomic data was used to interpret the LC/MS/MS data and to determinethe identity of the protein bands. The 50 kDa band had a strong matchwith ORF880. The Mascot score assigned to this match was 919, a highscore. Furthermore, ORF880 has a predicted molecular weight of 48,367Da. The nucleotide and amino acid sequences of orf880 are presented inFIGS. 41A and 41B, respectively.

Example 25. Heterologous Expression of Jeotgalicoccus ATCC 8456 Orf880in E. coli

Jeotgalicoccus ATCC 8456 Orf880 was identified as one of the two majorproteins in a highly purified enzyme fraction that catalyzed theconversion of free fatty acids to α-olefins. The genomic DNA encodingATCC 8456_orf880 was cloned into pCDF-Duet1 under the control of the T7promoter, and E. coli was transformed with various vectors, as describedbelow. The E. coli cells were cultured and the hydrocarbons produced bythe cells were analyzed as described in Example 23. When 0.05% stearicacid was fed to cultures of E. coli transformed with the8456_orf880-containing vector, the expression of 8456_orf880 led to theformation of 1-heptadecene in E. coli (see FIG. 42, which depicts GC/MStraces of α-olefins from E. coli either without (black) or with (gray)8456_orf880 expression). In contrast, adding 0.05% stearic acid tocultures of E. coli transformed with a vector control (not containingATCC_orf880) did not result in the production of 1-heptadecene. Thisdemonstrated that 8456_orf880 synthesized α-olefins from free fattyacids in an E. coli heterologous host. This result indicates thatα-olefin biosynthesis can be performed in heterologous organisms.Additionally, when E. coli cells expressing 8456_orf880 protein were fedwith 0.05% palmitic acid or 0.05% eicosanoic acid, the production of1-pentadecene or 1-nonadecene, respectively, was observed.

Example 26. In Vitro Synthesis of A-Olefins Using Orf880 HeterologouslyExpressed in and Purified from E. coli

The genomic DNA encoding ATCC8456_orf880 was cloned into the NdeI andXhoI sites of vector pET15b (Novagen) under the control of a T7 promoterfor expression in and purification from E. coli. This plasmid expressedan N-terminal His-tagged version of 8456_orf880.

An E. coli BL21 strain (DE3) (Invitrogen) was transformed withpET15b-ORF 880 using routine chemical transformation techniques. Proteinexpression was carried out by first inoculating a colony of the E. colistrain in 5 mL of LB media supplemented with 100 mg/L carbenecillin andshaken overnight at 37° C. to produce a starter culture. This starterculture was used to inoculate 1 L of an LB medium supplemented with 100mg/L carbenecillin. The culture was shaken at 37° C. until it reached anOD₆₀₀ value of 0.6. The culture was placed on ice for 10 minutes beforeIPTG was added to a final concentration of 250 μM. The culture was thenshaken at 18° C. for about 18 hours. The culture was then centrifuged at3,700 rpm for 20 minutes at 4° C. The pellet was resuspended in 30 mL ofbuffer containing 100 mM sodium phosphate buffer at pH 7.2, supplementedwith Bacterial ProteaseArrest (GBiosciences). The cells were sonicatedat 12 W on ice for 9 seconds with 1.5 seconds of sonication followed by1.5 seconds of rest. This procedure was repeated 5 times with 1 minuteintervals between each sonication cycle. The cell free extract wascentrifuged at 10,000 rpm for 30 minutes at 4° C. 5 mL of Ni-NTA(Qiagen) was added to the supernatant and the mixture was gently stirredat 4° C. The slurry was passed through a column to remove the resin fromthe lysate. The resin was then washed with 30 mL of buffer containing100 mM sodium phosphate buffer at pH 7.2, and 30 mM imidazole. Finally,the protein was eluted with 15 mL of 100 mM sodium phosphate buffer atpH 7.2 plus 250 mM imidazole. The protein solution was dialyzed with 200volumes of 100 mM sodium phosphate buffer at pH 7.2. Proteinconcentration was determined using the Bradford assay (Bio-Rad). 125μg/mL of protein was obtained.

To assay the in vitro fatty acid substrate specificity of ORF880,potassium salts of the following fatty acids were prepared:tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoicacid, and behenic acid (Sigma). The fatty acid solutions were made with2% ethanol and 2% Tergitol solution (Sigma, St. Louis, Mo.) to a finalconcentration of 20 mM.

The kinetics of the decarboxylation reaction and production wasdetermined. A 200 μl reaction mixture was prepared containing thefollowing reactants: 1.25 μM of ORF880, 200 μM of potassiumoctadecanoate, 200 μl dithiothreitol, and 100 mM sodium phosphate bufferat pH 7.2. The reaction mixture was incubated at room temperature andtime points were taken in duplicate between 5 minute and 120 minute. Thereaction was quenched and extracted by adding 100 μl of ethyl acetatecontaining 1-octadecene at 5 mg/L as an internal reference. Samples wereanalyzed using GC/MS using the alkane 1 splitless method, using thefollowing parameters: run time: 20 min; column: HP-5-MS Part No.19091S-433E (length of 30 meters; I.D.: 0.25 mm narrowbore; film: 0.25μM); sample: standard ethyl acetate extraction; inject: 1 μl Agilent6850 inlet; inlet: 300° C. splitless; carrier gas: helium; flow: 1.3mL/min; oven temp: 100° C. hold 5 min, 320 at 20° C./min, 320 hold 5min; det: Agilent 5975B VL MSD; det. temp: 300° C.; scan: 50-500 M/Z.Calibration curves were generated using 1-heptadecene dissolved in ethylacetate. Based upon this analysis, the product production was determinedto be linear from 5 minute to 60 minute.

To assay the reaction rates of different fatty acid substrates, thefollowing 200 μl reaction mixtures were prepared: 1.0 μM ORF 880 enzyme,200 μM of a test fatty acid salt, 200 μL dithiothreitol, and 100 mMsodium phosphate buffer at pH 7.2. The reactions were carried out atroom temperature and time points were taken in triplicates at 20 minuteand 47 minute using the extraction and analysis procedures as describedabove. Reference curves were generated using available chemicalstandards. In some instances, the chemical standards were not available.Under those circumstances, for example, cis-9-heneicosene was used as areference for 1-heneicosene, and 9-tricosene was used as a reference for1-tricosene. Activities were calculated by taking the difference betweenthe average α-olefin concentrations for each substrate at 47 minute and20 minute and then dividing the difference by 27 minute. The results aresummarized in Table 20.

TABLE 20 Activity of ORF880 with different fatty acid substratesActivity (nM alkene Substrate produced/min) tetradecanoic acid 22.9hexadecanoic acid 181.9 octadecanoic acid 77.2 eicosanoic acid 19.7behenic acid 30.6

These results demonstrate that heterologously expressed ORF880 was ableto convert fatty acid substrates to olefins in vitro. These data alsoshow that ORF880 had greater activity when hexadecanoic acid was thefatty acid substrate.

Example 27. Production of α-Olefins from Glucose by HeterologousExpression of Jeotgalicoccus ATCC 8456 ORF880 in E. coli MG1655 ΔFadD

1. Construction of fadD Deletion Strain

The fadD gene of E. coli MG1655 was deleted using the lambda red system(Datsenko et al., Proc. Natl. Acad. Sci. USA. 97: 6640-6645, 2000) asfollows:

The chloramphenicol acetyltransferase gene from pKD3 was amplified usingthe primers

fad1: (SEQ ID NO: 43)5′-TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATTGTCCACTTTGTGTAGGCTGGAGCTGCTTCG-3′; and fad2: (SEQ ID NO: 44)5′-CATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTGAAGAATTGCATATGAATATCCTCCTTTAGTTCC-3′.This PCR product was electroporated into E. coli MG1655 (pKD46). Thecells were plated on L-chloramphenicol (30 μg/mL)(L-Cm) and culturedovernight at 37° C. Individual colonies were selected and plated ontoanother L-Cm plate and cultured at 42° C. These colonies were thenpatched to L-Cm and L-carbenicillin (100 mg/mL) (L-Cb) plates andcultured at 37° C. overnight. Colonies that were Cm^(R) and Cb^(S) wereevaluated further by PCR to ensure the PCR product inserted at thecorrect site. PCR verification was performed on colony lysates of thesebacteria using primers fadF: 5′-CGTCCGTGGTAATCATTTGG-3′(SEQ ID NO:45);and fadR: 5′-TCGCAACCTTTTCGTTGG-3′(SEQ ID NO:46). Expected size of theΔfadD::Cm deletion was about 1200 bp (FIG. 10). The chloramphenicolresistance gene was eliminated using a FLP helper plasmid as describedin Datsenko et al. Proc. Natl. Acad. Sci. USA. 97: 6640-6645, 2000. PCRverification of the deletion was performed with primers fadF and fadR.The MG1655 ΔfadD strain was unable to grow on M9+oleate agar plates(oleate as carbon source). It was also unable to grow in M9+oleateliquid media.

2. Expression of Jeotgalicoccus ATCC 8456 orf880 in E. coli MG1655 ΔfadD

The genomic DNA encoding ATCC 8456_orf880, which was codon-optimized forexpression in E. coli, was cloned into vector OP80 (pCL1920 derivative)under the control of a P_(trc) promoter, and E. coli MG1655 ΔfadD wastransformed with the resulting vector. The E. coli cells were culturedat 37° C. in an M9 mineral medium supplemented with 20 μg/mL uracil and100 μg/mL spectinomycin. Glucose (1%, w/v) was the only source of carbonand energy. When the culture reached an OD₆₀₀ of 0.8 to 1.0, IPTG (1 mM)was added and the temperature was shifted to 25° C. After growth for anadditional 18 to 24 hours at 25° C., cells from 10 mL of culture werepelleted, resuspended in 1 mL methanol, sonicated for 30 minutes, andextracted with 4 mL hexane. After solvent evaporation, samples wereresuspended in 0.1 mL hexane and analyzed by GC-MS. In contrast to thevector-only control, E. coli cells transformed with the orf880-bearingvector produced the α-olefins 1-pentadecene and heptadecadiene. Thisresult indicates that expression of ORF880 confers the ability tobiosynthesize α-olefins to E. coli when cultured on glucose, and thatthe direct precursors are the most abundant fatty acids in E. coli,namely hexadecanoic acid and vaccenic acid (11-cis-octadecenoic acid).

Example 28. Identification of Carboxylic Acid Reductase (CAR) Homologs

The carboxylic acid reductase (CAR) from Nocardia sp. strain NRRL 5646can reduce carboxylic acids into corresponding aldehydes withoutseparate activating enzymes, such as acyl-CoA synthases (Li et al., J.Bacteriol. 179:3482-3487, 1997; He et al., Appl. Environ. Microbiol.70:1874-1881, 2004)). A BLAST search using the NRRL 5646 CAR amino acidsequence (Genpept Accession No. AAR91681) as the query sequenceidentified about 20 homologous sequences. Three homologs, listed inTable 21, were evaluated for their ability to convert fatty acids intofatty aldehydes in vivo when expressed in E. coli. At the nucleotidesequence level, carA, carB, and fadD9 (demonstrated 62.6%, 49.4%, and60.5% homology, respectively, to the car gene (AY495697) of Nocardia sp.NRRL 5646. At the amino acid level, CARA, CARB, and FadD9 demonstrated62.4%, 59.1% and 60.7% identity, respectively, to CAR of Nocardia sp.NRRL 5646.

TABLE 21 CAR-like Protein and the corresponding coding sequences.Locus_(—) Gene Genpept Accession tag Annotation in GenBank nameNP_217106 Rv 2590 Probable fatty-acid-CoA fadD9 ligase (FadD9) ABK75684MSMEG NAD dependent epimerase/ carA 2956 dehydratase family proteinYP_889972.1 MSMEG NAD dependent epimerase/ carB 5739 dehydratase familyprotein

Example 29. Expression of Car Homologs in E. coli

1. Plasmid Construction

Three E. coli expression plasmids were constructed to express the genesencoding the CAR homologs listed in Table 22, below. First, fadD9 wasamplified from genomic DNA of Mycobacterium tuberculosis H37Rv (obtainedfrom The University of British Columbia, and Vancouver, BC Canada) usingthe primers fadD9F and FadDR (see Table 22). The PCR product was firstcloned into PCR-blunt (Invitrogen) and then released as an NdeI-AvrIIfragment. The NdeI-AvrII fragment was then cloned between the NdeI andAvrII sites of pACYCDuet-1 (Novogen) to generate pACYCDuet-1-fadD9.

The carA and carB genes were amplified from the genomic DNA ofMycobacterium smegmatis MC2 155 (obtained from the ATCC (ATCC 23037D-5))using primers CARMCaF and CARMCaR or CARMCbF and CARMCbR, respectively(see, Table 22). Each PCR product was first cloned into PCR-blunt andthen released as an NdeI-AvrII fragment. Each of the two fragments wasthen subcloned between the NdeI and AvrII sites of pACYCDuet-1 (Novogen)to generate pACYCDUET-carA and pACYCDUET-carB.

TABLE 22 Primers used to amplify genes encoding CAR homologs fadD9FCAT ATGTCGATCAACGATCAGCGACTGAC (SEQ ID NO:47) fadD9RCCTAGG TCACAGCAGCCCGAGCAGTC (SEQ ID NO: 48) CARMCaFCAT ATGACGATCGAAACGCG (SEQ ID NO: 49) CARMCaRCCTAGG TTACAGCAATCCGAGCATCT (SEQ ID NO: 50) CARMCbFCAT ATGACCAGCGATGTTCAC (SEQ ID NO: 51) CARMCbRCCTAGG TCAGATCAGACCGAACTCACG (SEQ ID NO: 52)

2. Evaluation of Fatty Aldehyde Production

Plasmids encoding the CAR homologs (pACYCDUET-fadD9, pACYCDUET-carA, andpACYCDUET-carB) were separately co-transformed into the E. coli strainC41 (DE3, ΔfadE) (described in PCT/US08/058788) together withpETDuet-1-'TesA (described in PCT/US08/058788, the disclosures of whichis incorporated by reference herein).

The E. coli transformants were cultured in 3 mL of an LB mediumsupplemented with carbenicillin (100 mg/L) and chloramphenicol (34 mg/L)at 37° C. After overnight growth, 15 μl of culture was transferred into2 mL of a fresh LB medium supplemented with carbenicillin andchloramphenicol. After 3.5 hours of growth, 2 mL of culture weretransferred into a 125 mL flask containing 20 mL of an M9 medium with 2%glucose and with carbenicillin and chloramphenicol. When the OD₆₀₀ ofthe culture reached 0.9, 1 mM of IPTG was added to each flask. After 20hours of growth at 37° C., 20 mL of ethyl acetate (with 1% of aceticacid, v/v) was added to each flask to extract the organic compoundsproduced during the fermentation. The crude ethyl acetate extract wasdirectly analyzed with GC/MS as described below. The co-expression ofthe leaderless 'TesA and any of the three car genes in E. coli resultedin detectable fatty aldehyde production. In one fermentation,LS9001/pACYCDUET carB+pETDuet-1-'TesA produced an average of 120 mg/L offatty aldehydes. The retention times were 6.959 minutes for dodecanal,8.247 minutes for 7-tetradecenal, 8.37 minutes for tetradecanal, 9.433minutes for 9-hexadecenal, 9.545 minutes for hexadecanal, and 10.945minutes for 11-octadecenal. The presence of large amounts of fattyaldehydes is consistent with CAR being an aldehyde-generating, fattyacid reductase (AFAR). This mechanism is different from thealcohol-generating fatty acyl-CoA reductases (FAR), for example, JjFAR,and fatty acyl-CoA reductases, such as Acrl.

3. Substrate Preferences of the CAR Homologs

Distinct substrate preferences were observed among the three CARhomologs evaluated. FadD9 exhibited a strong preference for C₁₂ fattyacids relative to other fatty acids with carbon chain lengths greaterthan 12. Both CarA and CarB demonstrated wider substrate ranges thanFadD9.

4. Quantification and Identification of Fatty Aldehydes

A GC-MS experiment was performed using an Agilent 5975B MSD systemequipped with a 30 m×0.25 mm (0.10 μm film) DB-5 column. The columntemperature was 3-minute isothermal at 100° C. The column was programmedto rise from 100° C. to 320° C. at a rate of 20° C./min. When the finaltemperature was reached, the column remained isothermal for 5 minutes at320° C. The injection volume was 1 μL. The carrier gas, helium, wasreleased at 1.3 mL/min. The mass spectrometer was equipped with anelectron impact ionization source. The ionization source temperature wasset at 300° C.

Prior to quantification, various aldehydes were identified using twomethods. First, the GC retention time of each compound was compared tothe retention time of a known standard, such as laurylaldehyde(dodecanal). Second, identification of each compound was confirmed bymatching the compound's mass spectrum to a standard's mass spectrum inthe mass spectra library.

Example 30. Production of Fatty Alcohol by Heterologous Expression ofCar Homologs in E. coli Mg1655 (De3, ΔfadD)

1. Construction of fadD Deletion Strain

The fadD gene of E. coli MG1655 was deleted using the lambda red system(Datsenko et al., PNAS (USA). 97: 6640-6645, 2000) as follows: Thechloramphenicol acetyltransferase gene from pKD3 was amplified withprimers fad1:5′-TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATTGTCCACTTTGTGTAGGCTGGAGCTGCTTCG-3′(SEQ ID NO:43); and fad2:5′-CATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTGAAGAATTGCATATGAATATCCTCCTTTAGTTCC-3′(SEQ ID NO:44). This PCR product was electroporatedinto E. coli MG1655 (pKD46). The cells were plated on L-chloramphenicol(30 μg/mL) (L-Cm) and cultured overnight at 37° C. Individual colonieswere selected and plated onto another L-Cm plate and cultured at 42° C.These colonies were then patched to L-Cm and L-carbenicillin (100 mg/mL)(L-Cb) plates and cultured at 37° C. overnight. Colonies that wereCm^(R) and Cb^(S) were evaluated further by PCR to ensure the PCRproduct inserted at the correct site. PCR verification was performed oncolony lysates of these bacteria using primers fadF:5′-CGTCCGTGGTAATCATTTGG-3″(SEQ ID NO:45); and fadR:5′-TCGCAACCTTTTCGTTGG-3′(SEQ ID NO:46). Expected size of the ΔfadD::Cmdeletion was about 1200 bp. The chloramphenicol resistance gene waseliminated using a FLP helper plasmid as described in Datsenko et al.,Proc. Natl. Acad. Sci. USA, 97:6640-6645, 2000. PCR verification of thedeletion was performed using primers fadF and fadR. The MG1655 ΔfadDstrain was unable to grow on M9+oleate agar plates (using oleate ascarbon source). It was also unable to grow in M9+oleate liquid media.The growth defect was complemented by an E. coli fadD gene supplied intrans (in pCL1920-Ptrc).

2. Construction of MG1655(DE3, ΔfadD) Strain

To generate a T7-responsive strain, the 2 DE3 Lysogenization Kit(Novagen) was utilized, which is designed for site-specific integrationof 2DE3 prophage into an E. coli host chromosome, such that thelysogenized host can be used to express target genes cloned in T7expression vectors. λDE3 is a recombinant phage carrying the cloned genefor T7 RNA polymerase under lacUV5 control. Briefly, the host strain wascultured in an LB medium supplemented with 0.2% maltose, 10 mM MgSO₄,and antibiotics at 37° C., to an OD₆₀₀ of 0.5. Next, 10⁸ pfu 2 DE3, 10⁸pfu Helper Phage, and 10⁸ pfu Selection Phage were incubated with 10 μlhost cells. The host/phage mixture was incubated at 37° C. for 20minutes to allow the phage to be adsorbed into the host. Finally, themixture was pipetted onto an LB plate supplemented with antibiotics. Themixture was spread evenly using plating beads, and the plates wereinverted plates and incubated at 37° C. overnight.

λDE3 lysogen candidates were evaluated for their ability to support thegrowth of the T7 Tester Phage. T7 Tester Phage is a T7 phage deletionmutant that is completely defective unless active T7 RNA polymerase isprovided by the host cell. The T7 Tester Phage makes very large plaqueson authentic λDE3 lysogens in the presence of IPTG, while much smallerplaques are observed in the absence of inducer. The relative size of theplaques in the absence of IPTG is an indication of the basal levelexpression of T7 RNA polymerase in the lysogen, and can vary widelybetween different host cell backgrounds.

The following procedure was used to determine the presence of DE3lysogeny. First, candidate colonies were cultured in LB mediasupplemented with 0.2% maltose, 10 mM MgSO₄, and antibiotics at 37° C.,to an OD₆₀₀ of 0.5. An aliquot of T7 Tester Phage was then diluted in 1XPhage Dilution Buffer to a titer of 2×10³ pfu/mL. In duplicate tubes,100 μl host cells were mixed with 100 μL diluted phage. The host/phagemixture was incubated at room temperature for 10 minutes to allow thephage to be adsorb into the host. Next, 3 mL of molten top agarose wasadded to each tube containing host and phage. The contents of oneduplicate were plated onto an LB plate and the other duplicate onto anLB plate supplemented with 0.4 mM IPTG(isopropyl-b-thiogalactopyranoside) to evaluate induction of T7 RNApolymerase. Plates were allowed to sit undisturbed for 5 minutes untilthe top agarose hardened. The plates were then inverted at 30° C.overnight.

3. Construction of MG1655(DE3, ΔfadD, yjgB::kan) Strain

The yjgB knockout strain, MG1655(DE3, ΔfadD, yjgB::kan), was constructedusing the following the protocol of the lambda red system (Datsenko etal., Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000):

The kanamycin resistant gene from pKD13 was amplified with primersyjgBRn: 5′-GCGCCTCAGATCAGCGCTGCGAATGATTTTCAAAAATCGGCTTTCAACACTGTAGGCTGGAGCTGCTTCG-3′SEQ ID NO:53); and yjgBFn:5′-CTGCCATGCTCTACACTTCCCAAACAACACCAGAGAAGGACCAAAAAATGATTCCGGGGATCCGTCGACC-3′(SEQ ID NO:54). The PCR product was then electroporatedinto E. coli MG1655 (DE3, ΔfadD)/pKD46. The cells were plated onkanamycin (50 μg/mL) (L-Kan) and cultured overnight at 37° C. Individualcolonies were selected and plated onto another L-Kan plate and culturedat 42° C. These colonies were then patched to L-Kan and carbenicillin(100 mg/mL) (L-Cb) plates and cultured at 37° C. overnight. Coloniesthat were kan^(R) and Cb^(s) were evaluated further by PCR to ensure thePCR product was inserted at the correct site. PCR verification wasperformed on colony lysates of these bacteria using primers BF:5′-GTGCTGGCGATACGACAAAACA-3′(SEQ ID NO:55); and BR:5′-CCCCGCCCTGCCATGCTCTACAC-3′(SEQ ID NO:56). The expected size of theyjgB::kan knockout was about 1450 bp.

4. Evaluation of FadD on Fatty Alcohol Production Using MG1655 (DE3,ΔfadD) Strain

In Example 2, a fadE deletion strain was used for fatty aldehyde andfatty alcohol production from 'TesA, CAR homologs, and endogenousalcohol dehydrogenase(s) in E. coli. To demonstrate that CAR homologsused fatty acids instead of acyl-CoA as a substrate, the gene encodingfor acyl-CoA synthase in E. coli (fadD) was deleted so that the fattyacids produced were not activated with CoA. E. coli strain MG1655 (DE3,ΔfadD) was transformed with pETDuet-1-'TesA and pACYCDuet-1-carB. Thetransformants were evaluated for fatty alcohol production using themethods described herein. These transformants produced about 360 mg/L offatty alcohols (dodecanol, dodecenol, tetredecanol, tetredecenol, cetyl,hexadecenol, and octadecenol).

YjgB is an alcohol dehydrogenase. To confirm that YjgB was an alcoholdehydrogenase responsible for converting fatty aldehydes into theircorresponding fatty alcohols, pETDuet-1-'TesA and pACYCDuet-1-fadD9 wereco-transformed into either MG1655(DE3, ΔfadD) or MG1655(DE3, ΔfadD,yjgB::kan). At the same time, MG1655(DE3, ΔfadD, yjgB::kan) wastransformed with both pETDuet-1-'tesA-yjgB and pACYCDuet-1-fadD9.

The E. coli transformants were cultured in 3 mL of an LB mediumsupplemented with carbenicillin (100 mg/L) and chloramphenicol (34 mg/L)at 37° C. After overnight growth, 15 μL of culture was transferred into2 mL of a fresh LB medium supplemented with carbenicillin andchloramphenicol. After 3.5 hrs of growth, 2 mL of culture wastransferred into a 125 mL flask containing 20 mL of an M9 mediumcontaining 2% glucose, carbenicillin, and chloramphenicol. When theOD₆₀₀ of the culture reached 0.9, 1 mM of IPTG was added to each flask.After 20 hours of growth at 37° C., 20 mL of ethyl acetate (with 1% ofacetic acid, v/v) was added to each flask to extract the fatty alcoholsproduced during the fermentation. The crude ethyl acetate extract wasdirectly analyzed using GC/MS as described herein.

The yjgB knockout strain resulted in significant accumulation ofdodecanal and a lower fatty alcohol titer. The expression of yjgB fromplasmid pETDuet-1-'tesA-yjgB in the yjgB knockout strain effectivelyremoved the accumulation of dodecanal. The data indicated that YjgB wasinvolved in converting dodecanal into dodecanol and that there may beother alcohol dehydrogenase(s) present in E. coli to convert otheraldehydes into alcohols. Dodecanal accumulated in the yjgB knockoutstrain, but it was not observed in either the wild-type strain(MG1655(DE3, ΔfadD)) or the yjgB knockout strain with the yjgBexpression plasmid.

Example 31. Generation of 'TesA Library

In this Example, methods are described for preparing a mutant library of'TesA. A suitable expression vector such as pACYC-'TesA that encodes'TesA, the truncated TesA lacking a signal peptide, enables productionof the 'TesA protein in the host strain. The plasmid pACYC-'TesAincludes the 'tesA sequence under the regulation of a trc promoter, atranscription terminator, a p15a origin of replication, an open readingframe encoding laclq, and the beta-lactamase antibiotic resistance gene.

The 'TesA protein amino acid sequence is provided in FIG. 58 (SEQ IDNO:31).

The QuikChange Mutagenesis kit (Stratagene) enables the facileconstruction of large numbers of mutants. Use of this kit to constructeach 'TesA mutant starts with two complementary primers containing oneor more mismatched bases required to change the encoded amino acid atthe desired position. The primers are 25-45 nucleotides in length, withmelting temperature >78° C. as calculated using the formula:

T_(m)=81.5+0.41(% GC)675/N

where T_(m) is the melting temperature, % GC is the percent of residuesin the primer that are guanosine or cytidine, and N is the number ofnucleotides in the primer. For example, the primers:

(SEQ ID NO: 57) CACGTTATTGATTCTGGGTAATAGCCTGAGCGCCGGGTATCG and(SEQ ID NO: 58) CGATACCCGGCGCTCAGGCTATTACCCAGAATCAATAACGTGwere used to mutate the aspartic acid at residue 9 to asparagine, wherethe underlined bases indicate the codon that was changed.

The primers were used in a polymerase chain reaction with pACYC-'TesA asa template, using the following temperature cycling program: 1 minute at95° C.; followed by 18 cycles of 50 seconds at 95° C., 50 seconds at 60°C., and 5 minutes at 68° C.; and 7 minutes at 68° C. The reactionproducts were then digested using the restriction enzyme DpnI, toselectively degrade the methylated template DNA. The remaining DNA wasthen transformed into E. coli for isolation of plasmid clones, whichwere then sequenced to verify that the desired substitutions have beenobtained.

Example 32. Assays

In the following Examples, assays for determining protein content, freefatty acid levels, and hydrolysis of acyl-PNP and acyl-CoA substratesare described. Specific assays used herein are also set forth below.

1. Assay for Determination of Protein Content in Cell Lysates

Cell lysates of E. coli expression cultures producing 'TesA variantswere prepared for characterization. To generate the expression cultures,seed cultures were grown overnight at 37° C. in an LB medium containing1% (w/v) glucose and 100 μg/mL carbenicillin. The seed cultures werethen diluted 1:100 into the same medium and grown for 3 hours at 37° C.with shaking (200 rpm). A 40 μL aliquot of each culture was then addedto 360 μL of LS9-1 medium (described below) supplemented with 100 μg/mLcarbenicillin and grown in a 96-well culture plate. After 3 additionalhours of growth, isopropyl β-D-1-thiogalactopyranoside (IPTG, at 1 mMfinal concentration) and Bis-Tris Propane (pH 7.0, at 0.1 M finalconcentration) were added, and the cultures were allowed to growovernight.

Cell pellets were harvested by centrifugation of the expression cultures(10 minutes at 3,500 rpm). The growth medium is discarded and the cellpellets stored at −80° C. To prepare soluble extracts, the frozen cellpellets are lysed in 50% BugBuster (EMD Biosciences, Cat. No. 70584-4)in 25 mM sodium phosphate, pH 7.0. Following 40 minutes of agitation,the cell lysates are clarified by centrifugation (10 minutes at 3,500rpm). The concentration of protein in the supernatant of the cell lysateis then measured using the bicinchoninic acid (BCA) assay, according tothe protocol provided by manufacturer (Thermo Scientific, Cat. No.23225). The supernatant is then used in the assays described below.

Medium: 5x Salt Solution 1X final concentration Na₂HPO₄ 30 g 6 g/LKH₂PO₄ 15 g 3 g/L NaCl 2.5 g 0.5 g/L  NH₄Cl 5 g 1 g/L dH₂O to 1 L stocksolutions: final concentration: 10 mg/mL Thiamine (Vitamin B1) 1 mg/L 1MMgSO₄ 1 mM 1M CaCl₂ 0.1 mM 20% glucose 2.00% sterile water 20 mg/mLuracil 20 ug/mL high pH trace minerals 1000x 1x For 1 L LS9-1 media with1.0% glucose: 2 mL 5x Salt Soultion 100 μL Thiamine (B1) 1 ml MgSO₄ 100μL CaCl₂ 50 mL 20% Glucose 1 mL trace minerals 1 mL Uracil Water 1 L(premake it 750 mL) TM solution (filter sterilized): 27 g/L FeCl₃—6 H₂O2 g/L ZnCl₂—4H₂O 2 g/L CaCl₂—6H₂O 2 g/L Na₂MoO₄—2H₂O 1.9 g/L CuSO₄—5H₂O0.5 g/L H₃BO₃ 100 mL/L concentrated HCl q.s. w/Milli-Q water

2. Free Fatty Acid Analysis

'TesA variants are produced in E. coli expression cultures, and the freefatty acids produced by the cultures were analyzed. To generate theexpression cultures, seed cultures were first grown overnight at 37° C.in an LB medium containing 1% (w/v) glucose and 100 μg/mL carbenicillin,and then diluted 1:100 into the same medium and grown for 3 hours at 37°C. with shaking (200 rpm). 40 μL of each culture was then added to 360μL of LS9-1 medium supplemented with 100 μg/mL carbenicillin, and grownin a 96-well culture plate. After 3 additional hours of growth,isopropyl β-D-1-thiogalactopyranoside (IPTG, at 1 mM finalconcentration) and Bis-Tris Propane (pH 7.0, at 0.1 M finalconcentration) were added, and the cultures were allowed to growovernight.

The cultures were then acidified with 1 N HCl to a final pH of about 2.5and then extracted with 600 μL ethyl acetate. Free fatty acids in theorganic phase were derivatized with tetramethylammonium hydroxide (TMAH)to generate the respective methyl esters, which were then analyzed on agas chromatograph equipped with a flame ionization detector.

3. Fatty Acyl-PNP Hydrolysis Assay

In this assay system, the reagent solutions used were:

1. 2% Triton X-100 in 50 mM sodium phosphate, pH 7.0

2. 10 mM acyl-para-nitrophenol (acyl-PNP) in acetone

To prepare an acyl-PNP working solution, 600 μL acyl-PNP stock was addedto 9.4 mL phosphate buffer and mixed well.

The assay was performed by adding 40 μL of the acyl-PNP working solutionto each well of a 96-well plate, followed by the rapid addition of 40 μLof clarified cell lysate. The solutions were mixed for 15 seconds, andthe absorbance change was read at 405 nm in a microtiter plate reader at25° C. The esterase activity was expressed as the ratio of(AA405/sec)mut/(AA405/sec)wt, wherein (AA405/sec)mut was the change inabsorbance at 405 nm per second in samples containing mutant 'TesA, and(AA405/sec)_(wt) was the change in absorbance at 405 nm per second insamples containing wildtype 'TesA.

4. Acyl-CoA Hydrolysis Assay

In this assay system, the reagent solutions used were:

10 mM acyl-coenzyme A (acyl-CoA) in 50 mM sodium phosphate, pH 7.050 mM sodium phosphate, pH 8.0, 50 mM monobromobimane (MBB) (Novagen,Cat. No. 596105) in acetonitrile. To prepare acyl-CoA working solution,0.5 mL acyl-CoA stock and 0.5 mL MBB stock were added to 29 mL phosphatebuffer followed by mixing.

The assay was performed by adding 60 μL of the acyl-CoA working solutionto each well of a black 96-well plate, followed by the rapid addition of40 μL of clarified cell lysate. After mixing for 15 seconds, theprogress of the reaction was monitored by fluorescence (λ_(ex)=380 nm,λ_(em)=480 nm) in a microtiter plate reader at 25° C. The acyl-CoAthioesterase activity was expressed as the ratio of(ARFU/sec)mut/(ARFU/sec)_(wt), where (ARFU/sec)mut was the change inrelative fluorescence units per second in samples containing mutant'TesA, and (ARFU/sec)_(wt) was the change in relative fluorescence unitsper second in samples containing wildtype 'TesA.

5. Applying the Z Score Methodology

A Z-score determination was conducted following the Z score methodologyas follows.

The Z score for a sample is defined as the number of standard deviationsthe sample signal differs from the control population signal mean. The Zscore has been used to rank the mutants according to properties ofinterest such as, for example, substrate chain length specificity,relative preference for ester over thioester bonds, relative preferencefor thioester bonds over ester bonds, and the proportion or percentageof ester produced. The Z score is measured using the followingcalculation:

Z=(sample value−control average)/Standard deviation of controls

The positive control used to generate the mutant 'TseA library hereinwas wild type 'TesA.

In a normal distribution, about 2.1% of the data will comprise 2 or morestandard deviations above the mean, and about 0.1% of the data willcomprise 3 or more standard deviations above the mean. Therefore Zscores of 2 or greater, 3 or greater, −2 or less, −3 or less and soforth are used to define more and more stringent classes of data thatare unlikely to occur by random chance.

Those variants that have a Z score greater than 3 were marked as havingan improved performance in terms of preference for substrates of certainchain lengths and/or catalytic rate. Also, those variants that have a Zscore greater than 3 were marked, under other circumstances, asproviding an improved or enhanced proportional or percentage yield forfatty esters vs. free fatty acids. Additionally, those variants thathave a Z score of −3 or less were marked, in yet other circumstances, asproviding a reduced proportional or percentage yield for fatty estersvs. free fatty acids.

Substrate specificity numbers are defined as the kinetic slope of agiven mutant for one substrate, divided by the total of the kineticslopes for the three substrates studied in the PNP assay (C₁₀, C₁₂,C₁₄), where the kinetic slope is the observed initial rate for thehydrolysis of a given ester substrate.

For example, to calculate a substrate specificity number for C₁₀:

C₁₀ SubsSpec=Mutant Slope C₁₀/(Mutant Slope C₁₀+C₁₂+C₁₄)

Next a substrate specificity Z score was calculated. The Average andStandard Deviations of the substrate specificity numbers for thepositive controls were first calculated (for each plate), and thefollowing formula was applied:

Mutant C₁₀ SubSpec Z score=(Mutant SubSpec C₁₀−AvgSubSpec)/SDSubSpec

As another example, to calculate an ester specificity number:

EsterSpec=Mutant Slope C₁₄-PNP/Mutant Slope C₁₄-CoA

Next an ester specificity Z score was calculated. The Average andStandard Deviations of the ester specificity numbers for the positivecontrols were first calculated (for each plate), and the followingformula was applied:

Mutant Ester Specificity Z score=(MutantEsterSpec−AvgEsterSpec)/SDEsterSpec Those variants which have an EsterSpecificity Z score greater than 3 were defined and marked as having apreference for ester over thioester, and/or as having improved activity(i.e., catalytic rate) with regard to ester over thioester. Thosevariants which have an Ester Specificity Z score less than −3 weremarked as having a preference for thioester over ester.

Example 33. Free Fatty Acid Analysis of 'TesA Variants

In this Example, assay results identifying various properties of 'TesAvariants are provided. The analysis was conducted using the methodsdescribed above in Example 32. In the tables of FIGS. 45 and 46, themutations are presented using “Variant Codes,” each of which providesthe wildtype amino acid, followed by the position in the amino acidsequence, followed by the replacement amino acid (e.g., “S10A” indicatesthat the serine at position 10 in the amino acid sequence has beenreplaced by alanine in this particular variant).

Example 34. Analysis of 'TesA Variants

Assay results for 'TesA variants are provided in FIGS. 45 and 46. Theanalysis was conducted using the methods described above in Example 32.As shown in FIG. 45, activity levels on C₁₀, C₁₂ and C₁₄ substrates andsubstrate specificities were analyzed.

FIG. 45 depicts performance indices of certain 'TesA variants of themutant 'TesA library, which demonstrated improved performance comparedto the wildtype enzyme. FIG. 45A-B depict performance indices of 'TesAmutants in terms of specificity for substrates of certain chain lengths.

FIG. 46A depicts 'TesA mutants that provided increased or enhancedproportional or percentage yield of fatty esters vs. free fatty acids.FIG. 46B depicts 'TesA mutants that provided reduced proportional orpercentage yield of fatty esters vs. free fatty acids. Only mutants thathad Z scores above 3 are illustrated in the table and other mutantshaving lesser activity are not included. Notwithstanding thepresentation of data, it is submitted that a lower Z score may identifyvaluable mutants and the Z score cut-off of 3 provided in FIG. 45 is notintended to limit the scope of the invention.

The results are represented graphically along the entire length of the'TesA molecule in FIGS. 57A-C.

1. Fatty Acid Production Activity for 'TesA Variants

Assay results for fatty acid production activity in 'TesA variants areconducted using the methods described above in Example 32.

2. Fatty Acyl-PNP Assay of 'TesA Variants

Assay results for fatty acyl-PNP activity of 'TesA variants are providedin FIG. 45. The analysis was conducted using the methods described abovein Example 32.

3. Acyl-CoA Analysis of 'TesA Variants

Assay results for acyl-CoA activity of 'TesA variants are provided inFIG. 45. The analysis was conducted using the methods described above inExample 32.

4. Preference for Thioester (Acyl-CoA) over Ester (Acyl-PNP)

Assay results for acyl-CoA activity and acyl-PNP activity of 'TesAvariants are conducted using the methods described above in Example 32.

5. Preference for Ester (Acyl-CoA) over Thioester (Acyl-CoA)

Assay results for acyl-CoA activity and acyl-PNP activity of 'TesAvariants are conducted using the methods described above in Example 32.

Example 35. Direct Production of Fatty Esters in the Absence of EsterSynthase

In this example, the ability of 'TesA to catalyze thetransesterification of a fatty acyl-CoA into the corresponding fattyester in the presence of an alcohol in vitro is demonstrated. E. coli'TesA enzyme was recombinantly expressed and purified to homogeneity asan N-terminal 6× His-tagged protein. In particular, the TesA geneencoding thioesterase I enzyme from E. coli (SEQ ID NO:31 of FIG. 58)was inserted into a pET15-b vector (Novagen), which vector carried anN-terminal 6× His-tag, and transformed into BL21-DE3 cells forexpression. Cells were cultured in LB media at 37° C., 200 rpm, untilOD₆₀₀ reached 1.0, induced with 0.5 mM IPTG (final), and then allowed togrow at 28° C. for an additional 5 hours. After harvesting at 6,000 rpm,the pellet was resuspended in 40 mL of 100 mM Tris-HCl, pH 7.4,sonicated and centrifuged at 10,000 rpm for 20 minutes. Clarified lysatewas then applied to a His-bind column (Calbiochem) and the protein waspurified as per the manufacturer's instruction. Eluted protein was thendialyzed into a buffer containing 25 mM sodium phosphate, pH 7.2, and10% glycerol for storage and use. Thioesterase activity of the purified'TesA enzyme was determined.

Catalysis of fatty acyl-CoA to fatty ester by 'TesA involves anucleophilic attack by an alcohol on the carbonyl center subsequent tothe exit of the coenzyme A moiety from the active site. The rate ofspontaneous transesterification of palmitic acid by ethanol in theabsence of 'TesA was analyzed to prove that ethanol can replace water asthe nucleophile to form fatty esters instead of fatty acids.

Accordingly, a 4 mM (about 1 mg/mL) aliquot of palmitic acid (C₁₆—COOH)(Sigma) was incubated with varying amounts of ethanol for different timeperiods at room temperature. Samples were extracted with a 1:1volumetric ratio of ethyl acetate and the extract was analyzed usingGC-MS for the presence of ethyl palmitate. The results are compiled inTable 23 below, which indicated that spontaneous transesterificationbetween ethanol and palmitic acid occurs at a conversion rate of lessthan 0.01 mole/mole of palmitic acid.

TABLE 23 % Ethanol C₂C₁₆ formed*, % conversion % conversion (v/v) mg/L(g/g) (mole/mole) 0 0 0 0 20 0.34 0.034 0.030 30 0.25 0.025 0.022 400.25 0.025 0.022 50 0.35 0.035 0.031 *Average of two data points.

The rate of in vitro transesterification catalyzed by 'TesA onpalmitoyl-CoA substrate was analyzed. Reactions were carried out at roomtemperature for 1 hour in a buffer containing 100 μM of palmitoyl-CoA,100 μM of Phosphate buffer pH 7.0 and 1 mM BSA, either in the presenceor absence of 1.5 μM of purified 'TesA. Ethanol concentrations variedbetween 0-60% (v/v). 1:1 volumetric ratio of ethyl acetate was used forquenching and subsequent extraction. Formation of ethyl palmitate wasmonitored using GC-MS. Table 24 summarizes the results.

TABLE 24 Ethyl % Ethyl palmitate palmitate conversion % Ethanol (mg/L)formed (g/g of conversion % v/v −′TesA +′TesA (mg/L) C16-CoA)(mole/mole) 0 0 0 0 0 0 5 0 0 0 0 0 10 0 4.12 4.12 4.12 14.57 20 0 6.646.64 6.64 23.49 40 0 1.88 1.88 1.88 6.65 60 0 1.74 1.74 1.74 6.15

The results indicate that 'TesA thioesterases efficiently catalyzes thetransesterification of an acyl-CoA, palmitoyl-CoA, into ethyl palmitatein presence of ethanol. Maximum yield obtained was 23.5 mole/mole ofpalmitoyl-CoA. Given that yields for spontaneous conversion of palmiticacid to palmitic ester are extremely low compared to those in presenceof 'TesA (i.e., indicating a >1,000-fold increase) the conversion occursenzymatically. Based on our data, maximum transesterification yieldsoccurred at 10-20% ethanol (v/v) levels. Higher alcohol concentrationsaffect enzyme stability and/or activity adversely and therefore resultin lower ester yields.

From these results, a conclusion was reached that thioesterase cancatalyze the direct esterification of an acyl-CoA substrate in thepresence of alcohol. It will be possible to modify the ester product bychanging the alcohol (e.g., by using methanol, propanol or butanol)and/or the alcohol concentration.

Example 36. In Vivo Production of Fatty Esters by Thioesterase

In this example, the ability of 'TesA to produce esters in vivo in theabsence of heterologously expressed ester synthase was investigated.Ester formation in the absence of a heterologously expressed estersynthase was observed in the E. coli strain MG1655 (ΔfadE), which alsocarries an artificial operon containing 'tesA and fadD under the controlof a trc promoter, along with a kanamycin marker gene. The operon wasintegrated into the chromosome, interrupting the native lacZ gene. Thisstrain was tested in a shake flask fermentation using media comprising 6g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 1 mg/L thiamine, 1mM MgSO₄, 0.1 mM CaCl, supplemented with extra NH₄Cl (an additional 1g/L), Bis-Tris buffer (0.2 M), Triton X-100 (0.1% v/v), and traceminerals (27 mg/L FeCl₃-6 H₂O, 2 mg/L ZnCl₂-4H₂O, 2 mg/L CaCl₂-6H₂O, 2mg/L Na₂MoO₄-2H₂O, 1.9 mg/L CuSO₄-5H₂O, 0.5 mg/L H₃BO₃, 100 mL/Lconcentrated HCl).

An LB+antibiotics pre-seed culture was inoculated with a scraping from aglycerol stock or from a single colony. It was cultured for 6 to 8 hoursuntil the OD₆₀₀ reached >1.0. A fermentation medium plus 2% glucose(w/v)+antibiotics overnight seed culture was inoculated with the LBpre-seed culture to 4% (v/v). 15 mL fermentation media+3% glucose(w/v)+antibiotics production cultures were prepared in 125 mL baffledshake flasks. An appropriate amount of the overnight seed culture wasused to inoculate the production culture such that the starting OD₆₀₀ inthe production culture flask was about 0.5. The flasks were allowed togrow until the OD₆₀₀ therein reached 1.0, at which point the cultureswere induced with 1 mM IPTG (final concentration) and fed methanol orethanol (at 2% v/v). The fermentation runs were allowed to continue forthe indicated amount of time post-induction. All culture steps wereperformed at 32° C. with shaking at 200 rpm.

Whole broth extractions were performed using a standard microextractionprocedure. In brief, 500 μl of broth was transferred to amicrocentrifuge tube, to which 100 μl of 1M HCl was added. The acidifiedcultures were extracted with 500 μl of ethyl acetate, vortexed for 5minutes, and centrifuged at top speed for 1 minute. The organic layerwas analyzed using GC-FID for both simultaneous fatty acid methyl ester(FAME) and free fatty acid (FFA) quantification and simultaneous fattyacid ethyl ester (FAEE) and FFA quantification.

In samples containing FAEE and FFA, the FFA were derivatized withBis(trimethylsilyl)trifluoroacetamide before quantification.

The MG1655 (ΔfadE) pTrc-'TesA_fadD strain, which was cultured and fed 2%methanol at induction, produced 2 g/L total FAMEs by the 24 hour timepoint and 3.5 g/L total FAMEs by the end of the fermentation at 48 hours(FIG. 48). Minimal amounts of FFAs were detected, about 100 mg/L intotal. The cultures reached their highest density, OD₆₀₀ about 11, after24 hours and did not continue to grow in the following 24 hours.Specific productivity was calculated to be about 200 mg/L/OD at 24hours, and about 300 mg/L/OD at 48 hours. These data indicated that,with the overexpression of 'tesA and fadD, even in the absence of a waxsynthase, FAME production was observed.

To assess the ability of FadD or 'TesA to independently produce FAME, asecond fermentation was carried out testing two different E. colistrains carrying plasmids with either fadD, 'tesA, or both fadD and'tesA. The plasmids were all pACYC-based and expression was driven by atrc promoter. Three different MG1655 (ΔfadE) strains were tested, onewith a fadD only plasmid, one with a 'tesA only plasmid, and one with'tesA and fadD with 'tesA being located upstream of fadD. Two C41(ΔfadE) were tested, both carrying 'tesA and fadD, but with the genes indifferent order relative to the promoter. These strains were cultured inthe media described above and fed 2% methanol at induction and grown foran additional 25 hours post-induction. The strain expressing only fadDdid not produce any FAMEs while the 'tesA strain produced only about 150mg/L FAMEs (FIG. 49). Having both 'TesA and fadD improves upon FAMEproduction over 'TesA alone. The two C41 strains produced a furtherincrease in FAME production, as observed in the strain carrying aplasmid in which fadD is upstream of 'tesA, over the strain expressing'tesA and fadD in the opposite order. This suggested that higher FadDexpression enhanced the ability of 'TesA to produce esters. Since 'TesAcan cleave both acyl-ACPs and acyl-COAs, it is likely that theproduction of acyl-COAs by FadD is allowing for the FFAs generated by'TesA to be recycled back to the thioesterase to either be convertedback into FFAs by hydrolysis or taken all the way to FAMEs byalcoholysis. Examination of the FFA titers leads to the conclusion thatonly the strain expressing 'TesA produced significant amounts of FFA,while the strains expressing fadD produced very little FFA (FIG. 50).

'TesA was tested for its ability to utilize ethanol for the directformation of fatty acid ethyl ester (FAEE). The two MG1655 (ΔfadE)strains from the experiment described above, the fadD overexpressionstrain and the 'TesA overexpression strain, were tested. Also includedin this experiment was the MG1655 (ΔfadE) with the integrated 'tesAjadDoperon under the control of a trc promoter. All strains were culturedusing the protocol described above. At induction, all strains were fed2% (v/v) of methanol or 2% (v/v) of ethanol. In addition, the MG1655(ΔfadE)+fadD strain was fed 0.05% (w/v) of C_(14:0) fatty acid to ensurethat sufficient free fatty acid substrate was available to FadD forcatalyzing the potential alcoholysis reaction. The fermentations wereallowed to continue for 24 hours.

Under these fermentation conditions, FadD alone was again unable toproduce the requisite C₁C₁₄:0 FAME or the C₂C₁₄:0 FAEE, indicating thatFadD was not sufficient for ester formation (FIG. 51). However, 'TesAalone was able to produce FAEEs and as before, overexpression of 'tesAand fadD boosts overall production of FAEEs over having 'tesA alone.While overall FAEE titers were lower than FAME titers, this datademonstrate that 'TesA can also use ethanol in addition to methanol forthe formation of fatty esters. Analysis of FFA formation under thesefermentation conditions indicates that the strains behaved similarlywith ethanol feeding as they did with methanol feeding (FIG. 52).

The FFA present in the fadD samples was contributed almost entirely bythe C₁₄:0 FFA fed during fermentation. The strain expressing 'tesAproduced a large amount of FFA, while the strain expressing 'tesA andfadD showed very little accumulation of FFA. In the presence of 'TesA,only 14% conversion of FFA to FAME or a 2.3% of FFA to FAEE wasobserved. In the presence of 'TesA and FadD, nearly a 100% conversion ofFFA to either FAME or FAEE was observed. These data suggest that 'TesAis necessary and sufficient for fatty acid alcohol ester formation, butthe overexpression of FadD along with 'TesA is important for increasedFAME and FAEE formation.

The previous results suggest that E. coli 'TesA can produce FAME andFAEE when fed the appropriate alcohols during fermentation. To determinewhether this is a function unique to E. coli 'TesA, the ability of otherheterologously expressed thioesterases to produce FAMEs wasinvestigated. 'TesA homologs from Photorhabdus luminescens and Vibrioharveyi along with a TesB from Photobacterium profundum wereoverexpressed from pACYC-based plasmids in the strain MG1655 (ΔfadE) andtested alongside the E. coli 'TesA overexpression strain from theprevious fermentations. Shake flask fermentations were carried out infermentation media and allowed to continue for 24 hours post-induction.The results indicated that the two 'TesA homologs were also able togenerate FAMEs (FIG. 53). P. luminescens 'TesA produced FAME at a levelcomparable to E. coli 'TesA, while the V. harveyi 'TesA was able toproduce much more FAME than E. coli 'TesA. When looking at the FFAtiters, the P. luminescens 'TesA produced less FFA than E. coli 'TesA,but again, the V. harveyi 'TesA produced much larger FFA titers whencompared to its E. coli counterpart (FIG. 54). Interestingly, the V.harveyi 'TesA was highly active and was able to produce higher FAME andFFA titers than the control strain expressing E. coli 'TesA; moreover,its FFA to FAME conversion rate was over 30% to E. coli 'TesA's 14%.Additionally, despite producing lower total FAME titers, the strainexpressing P. luminescens 'TesA showed that FAME constituted over 60% ofthe total FAME+FFA titer.

1. Ester Synthase Activity in Other 'TesA Homologs

The 'TesA homologs from Escherichia coli, Pectobacterium atrosepticum,Photobacterium profundum, Photorhabdus luminescens, Pseudomonas putida,and Vibrio harveyi were cloned into the expression vector pACYC underthe control of a trc promoter. All sequences were cloned as truncatedgenes lacking a signal peptide sequence, in order to achieve cytoplasmicexpression. DNA and amino acid sequences for the homologs are shown inTable 26. An alignment of the amino acid sequences is shown in Table 27.

The plasmids were transformed into E. coli MG1655 ΔfadE and culturedovernight at 37° C. on LB agar plates containing 100 μg/mLcarbenicillin. Individual colonies were selected and cultured at 37° C.in an LB broth containing 1% (w/v) glucose and 100 μg/mL carbenicillinuntil OD₆₀₀ reached a value of about 1.0. 200 μL of the culture was thendiluted into 1.8 mL of an M9 medium containing 100 μg/mL carbenicillin.After growing the cultures for 3 hours at 37° C., IPTG (1 mM finalconcentration), as well as Bis-Tris Propane buffer (0.1 M, pH 7.0), andmethanol (2% v/v) were added.

After 20 hours of growth at 37° C., 1 mL of culture was extracted byadding 100 μL 1 N HCl and 250 μL ethyl acetate. A C20 free fatty acidinternal standard was included in the ethyl acetate solution.

The fatty acids and methyl esters were analyzed on a gas chromatographTrace GC Ultra (Thermo Electron Corp) equipped with a flame ionizationdetector. The total amount of fatty acid (FFA) and fatty acyl methylester (FAME) produced varied among the homologs studied (see FIG. 60).

E. coli 'TesA produced about 300 mg/L in total fatty products, while thePseudomonas putida homolog generated nearly 4 times that amount. Theproportion of FAME produced was also dependent on which 'TesA homologwas expressed. Whereas only 3% of total product generated by 'TesA fromPseudomonas putida was FAME, more than 25% of total product generated byVibrio harveyi 'TesA was FAME. These results indicate that esterformation is catalyzed and influenced by 'TesA, rather than being apurely chemical process that is not affected by the enzyme. It followsthat this activity is a function of the amino acid sequence of theenzyme and that it can be engineered to increase or decrease thepropensity for ester production.

To determine whether FadD overexpression would increase FAME titers, theplasmids were then transformed into E. coli MG1655 ΔfadE carrying thefadD gene on the pCL1920 plasmid, under the control of a trc promoter.The transformed cells were cultured overnight at 37° C. on LB agarplates containing 100 μg/mL carbenicillin and 100 μg/mL spectinomycin.Individual colonies were selected and cultured at 37° C. in LB brothcontaining 1% (w/v) glucose, 100 μg/mL carbenicillin, and 100 μg/mLspectinomycin until OD₆₀₀ reached a value of about 1.0. 200 μL of theculture was then diluted into 1.8 ml of an M9 medium containing 100μg/mL carbenicillin and 100 μg/mL spectinomycin. After growing thecultures for 3 hours at 37° C., IPTG (1 mM final concentration), as wellas Bis-Tris Propane buffer (0.1 M, pH 7.0) and methanol (2% v/v) wereadded.

After 20 hours of growth at 37° C., 1 ml of culture was extracted byadding 100 μL 1 N HCl and 250 μL ethyl acetate. A C20 free fatty acidinternal standard was included in the ethyl acetate solution.

The fatty acids and methyl esters were analyzed on a gas chromatographTrace GC Ultra (Thermo Electron Corp) equipped with a flame ionizationdetector. As observed previously with E. coli 'TesA, coexpression ofFadD increased the proportion of FAME produced for all homologs tested(See FIG. 61). Therefore, co-expression of an acyl-CoA synthase inconjunction with 'TesA homologs can be used to increase esterproduction. Interestingly, the total titer of FFA plus FAME produced by'TesA from P. putida was much lower when FadD was co-expressed. Thissuggests that P. putida 'TesA may be more specific for acyl-ACPsubstrates than acyl-COAs, and can be co-expressed with an estersynthase or other thioesterase with greater activity against acyl-COAsto further increase ester production.

2. Enhanced Ester Synthesis by a 'TesA Mutant

As mentioned above, the studies of 'TesA homologs have indicated thatester synthase activity in 'TesA in an engineerable trait; that is, onecan make changes in the amino acid sequence of the enzyme to improve theproduction of esters. To this end, a mutant of E. coli 'TesA wasconstructed with enhanced ester synthase activity. Replacing Ser10, thenucleophilic serine residue in the active site of 'TesA, with cysteineto generate the S10C mutant yields an improved 'TesA enzyme thatproduces a higher proportion of FAME.

Plasmids encoding wildtype E. coli 'TesA, the S10C mutant, or no 'TesAwere transformed into E. coli MG1655 ΔfadE and cultured overnight at 37°C. on LB agar plates containing 100 μg/mL carbenicillin. Individualcolonies were selected and cultured overnight at 37° C. in an LB brothcontaining 1% (w/v) glucose and 100 μg/ml carbenicillin. The cultureswere then diluted 1:100 in a fresh LB medium supplemented with 1% (w/v)glucose and 100 μg/mL carbenicillin, and cultured at 37° C. until OD₆₀₀reached a value of about 1.0. 200 μL of the culture was then dilutedinto 1.8 mL of an M9 medium containing 100 μg/mL carbenicillin. Aftergrowing the cultures for 3 hours at 37° C., IPTG (1 mM finalconcentration) was added, as well as Bis-Tris Propane buffer (0.1 M, pH7.0) and methanol (2% v/v).

After 20 hours of growth at 37° C., 1 mL of culture was extracted byadding 100 μL 1 N HCl and 250 μL ethyl acetate. A C20 free fatty acidinternal standard was included in the ethyl acetate solution.

The fatty acids and methyl esters were analyzed on a gas chromatographTrace GC Ultra (Thermo Electron Corp) equipped with a flame ionizationdetector. The total amount of fatty acid (FFA) and fatty acyl methylester (FAME) was greater in cultures of wildtype E. coli 'TesA (316mg/L) compared to the S10C mutant (136 mg/L), but the proportion of FAMEin S10C (47%) was greater than that observed with wildtype 'TesA (9%).This demonstrates that the sequence of 'TesA can be modified to affectthe proportion of esters produced (See FIG. 62).

TABLE 26 Sequences of ‘TesA homologs studied in Example 36 SpeciesDNA Sequence Amino Acid Sequence Escherichia ATGGCGGACACGTTATTGAMADTLLILGDSLSAGYRMSA coli TTCTGGGTGATAGCCTGAG SAAWPALLNDKWQSKTSVVCGCCGGGTATCGAATGTCT NASISGDTSQQGLARLPALLK GCCAGCGCGGCCTGGCCTGQHQPRWVLVELGGNDGLRG CCTTGTTGAATGATAAGTG FQPQQTEQTLRQILQDVKAAGCAGAGTAAAACGTCGGT NAEPLLMQIRLPANYGRRYN AGTTAATGCCAGCATCAGCEAFSAIYPKLAKEFDVPLLPF GGCGACACCTCGCAACAA FMEEVYLKPQWMQDDGIHPGGACTGGCGCGCCTTCCGG NRDAQPFIADWMAKQLQPL CTCTGCTGAAACAGCATCAVNHDS (SEQ ID NO: 31) GCCGCGTTGGGTGCTGGTT GAACTGGGCGGCAATGACGGTTTGCGTGGTTTTCAGC CACAGCAAACCGAGCAAA CGCTGCGCCAGATTTTGCAGGATGTCAAAGCCGCCAA CGCTGAACCATTGTTAATG CAAATACGTCTGCCTGCAAACTATGGTCGCCGTTATAA TGAAGCCTTTAGCGCCATT TACCCCAAACTCGCCAAAGAGTTTGATGTTCCGCTGCT GCCCTTTTTTATGGAAGAG GTCTACCTCAAGCCACAATGGATGCAGGATGACGGTA TTCATCCCAACCGCGACGC CCAGCCGTTTATTGCCGACTGGATGGCGAAGCAGTTGC AGCCTTTAGTAAATCATGA CTCATAA (SEQ ID NO: 32)Pectobacterium ATGGCTGATACATTATTAA MADTLLILGDSLSAGYQMPA atrosepticumTTCTGGGTGATAGCCTCAG ANAWPTLLNTQWQTQKKGI TGCGGGCTACCAGATGCCGAVVNASISGDTTAQGLARLP GCCGCTAACGCCTGGCCAA ALLKQHQPRWVLIELGGNDGCGCTGCTGAACACGCAGTG LRGFPAPNIEQDLAKIITLVK GCAGACGCAGAAAAAGGGQANAKPLLMQVRLPTNYGR CATCGCCGTGGTTAACGCC RYTESFSNIYPKLAEQFALPLAGCATTAGCGGCGACACC LPFFMEQVYLKPEWIMEDGI ACCGCACAGGGGCTGGCGHPTRDAQPFIAEWMAKQLEP CGACTTCCTGCCTTACTGA LVNHES (SEQ ID NO: 59)AACAACATCAGCCGCGTTG GGTGTTGATTGAACTGGGC GGCAATGACGGGCTTCGGGGGTTTCCGGCACCCAATA TCGAGCAGGATCTGGCGA AAATCATTACGCTAGTCAAACAGGCTAACGCTAAGCCT CTGCTGATGCAGGTTCGTT TGCCAACCAACTATGGCCGCCGCTACACCGAGTCATTC AGCAACATTTACCCCAAAC TCGCGGAGCAGTTTGCGCTTCCTCTGCTGCCTTTCTTTA TGGAGCAGGTGTATCTTAA ACCGGAGTGGATCATGGAAGATGGCATCCATCCAACC CGTGATGCCCAACCGTTTA TCGCAGAATGGATGGCGAAGCAGCTGGAACCCTTAGT TAACCATGAGTCTTAA (SEQ ID NO: 60) PhotobacteriumATGGGCAACACATTACTGG MAWGNTLLVVGDSLSAGYQ profundum TTGTCGGTGATAGCTTGAGMRAEQSWPVLLQPALKQQG CGCGGGCTATCAAATGCGG HEITVVNASISGDTTGNGLARGCAGAACAAAGCTGGCCG LPTLLQQHKPAYVIIELGAND GTGTTACTGCAACCCGCATGLRGFPQGTIRNNLSQMITEI TAAAGCAACAAGGTCACG QNADAKPMLVQIKVPPNYGAAATCACCGTTGTAAATGC KRYSDMFSSIYPQLSKELATP CAGTATTTCAGGCGATACALLPFFLEQIILKQEWMMNDG ACAGGAAACGGCTTGGCTC LHPKSDAQPWIAEYMAENIAGATTGCCTACATTATTACA PYL (SEQ ID NO: 61) ACAACATAAACCAGCTTACGTCATAATTGAACTCGGGG CGAATGATGGCTTACGTGG TTTCCCTCAAGGTACTATACGTAACAATCTCAGCCAAA TGATCACTGAAATTCAAAA TGCTGATGCCAAGCCAATGCTCGTGCAGATAAAAGTGC CGCCCAATTACGGCAAACG CTACAGTGATATGTTCAGTTCTATTTACCCTCAACTCA GTAAAGAGTTAGCCACACC ACTGTTACCTTTCTTTTTAGAGCAGATCATTTTAAAACA AGAATGGATGATGAATGA CGGTTTGCATCCTAAATCTGATGCTCAGCCATGGATTG CCGAATATATGGCTGAGAA TATCGCGCCTTATTTATAA(SEQ ID NO: 62) Photorhabdus ATGGCTGATACCCTTCTGA MADTLLILGDSLSAGYHLPIEluminescens TTCTCGGTGATAGCCTTAG QSWPALMEKKWQKSGNKIT TGCCGGTTACCATCTGCCTVINGSISGNTAAQGLERLPEL ATTGAGCAGTCATGGCCTG LKQHKPRWVLIELGANDGLRCTTTGATGGAAAAAAAGTG GFPPQHTEQDLQQIITLVKQA GCAAAAATCCGGCAATAANIQPLLMQIRLPPNYGRRYTE AATCACGGTCATCAACGGC SFAKIYPKLAEYNQIPLLPFYAGCATCAGCGGCAACACC MEQVAIKPEWVQQDGLHPN GCCGCTCAGGGCCTTGAGCLAAQPFIADWMSDTLSAHLN GGCTACCTGAATTACTTAA YS (SEQ ID NO: 63)ACAACATAAACCCCGTTGG GTACTGATAGAGCTGGGTG CCAACGATGGATTACGCGGTTTTCCTCCACAACACACC GAACAAGATCTACAACAG ATCATTACTTTAGTGAAACAAGCTAATATTCAGCCTTT ATTGATGCAGATCCGTCTA CCACCAAACTATGGGCGCCGTTATACCGAGTCTTTTGC CAAGATTTACCCCAAACTG GCAGAATATAATCAAATTCCCCTGCTCCCGTTTTATAT GGAGCAAGTCGCCATTAA ACCGGAGTGGGTGCAACAAGATGGGTTACATCCTAAT CTGGCAGCCCAACCATTTA TCGCCGATTGGATGTCTGACACACTATCAGCACATCTT AATTATTCTTAA (SEQ ID NO: 64) PseudomonasATGGCAGGAACACTGCTG MAGTLLVVGDSISAGFGLDS putida GTTGTTGGCGATAGTATCARQGWVSLLQARLRDEGFDD GCGCCGGTTTTGGCCTGGA KVVNASISGDTSAGGQARLPTAGCCGTCAGGGCTGGGTG ALLAAHKPSLVVLELGGNDG TCTCTCTTGCAGGCCCGTCLRGQPPAQLQQNLASMIERS TCAGGGACGAAGGTTTTGA RQAGAKVLLLGMRLPPNYGCGACAAAGTGGTCAATGCT VRYTTAFAKVYEQLAADKQ TCGATCAGTGGCGATACCAVPLVPFFLEGVGGVPELMQA GCGCAGGTGGCCAGGCGC DGIHPAQGAQQRLLENAWPGGCTGCCGGCGCTGCTTGC AIKPLL (SEQ ID NO: 65) AGCACATAAACCGAGCCTGGTGGTGCTGGAGCTGGGC GGCAACGATGGCCTGCGC GGGCAGCCGCCTGCACAATTGCAACAAAATCTTGCCTC GATGATCGAGCGTTCGCGT CAGGCAGGGGCCAAGGTGCTGCTATTGGGCATGCGCC TGCCGCCCAATTATGGTGT GCGTTACACCACCGCCTTTGCCAAGGTGTATGAACAG CTGGCAGCGGACAAACAG GTTCCCTTGGTGCCGTTTTTCCTCGAAGGGGTAGGGGG CGTACCTGAACTGATGCAG GCTGATGGCATCCATCCGGCCCAGGGGGCTCAGCAGC GCCTGCTGGAAAATGCCTG GCCAGCGATAAAACCCTTGCTGTGA (SEQ ID NO:66) Vibrio ATGAGCGAAAAGCTACTTG MSEKLLVLGDSLSAGYQMPIharveyi TTTTGGGCGACAGCCTGAG EESWPSLLPGALLEHGQDVK CGCTGGTTATCAAATGCCTVVNGSISGDTTGNGLARLPSL ATAGAGGAGAGTTGGCCT LEQHTPDLVLIELGANDGLRAGCTTACTCCCAGGCGCGT GFPPKLITLNLSKMITMIKDS TATTAGAACATGGCCAAGAGADVVMMQIRVPPNYGKRY TGTAAAAGTTGTAAACGGT SDMFYDIYPKLAEHQQVALAGCATCTCTGGTGACACCA MPFFLEHVIIKPEWMMDDGL CAGGCAATGGCCTTGCACGHPKPEAQPYIADFVAQELVK GTTACCTTCTCTCCTTGAG HL (SEQ ID NO: 67)CAACACACGCCCGATTTGG TACTGATTGAGCTTGGCGC TAACGATGGCCTACGCGGTTTCCCACCTAAACTTATTA CGTTAAACCTATCGAAAAT GATTACCATGATCAAAGATTCTGGTGCGGATGTCGTCA TGATGCAAATCCGCGTCCC ACCAAATTATGGTAAGCGTTACAGCGATATGTTCTACG ACATCTACCCTAAACTGGC AGAACATCAGCAAGTAGCGCTAATGCCGTTCTTCTTA GAGCATGTCATCATTAAAC CAGAATGGATGATGGACGATGGCTTGCACCCAAAACC GGAAGCTCAACCCTACATT GCTGACTTTGTCGCTCAAGAATTGGTTAAACATCTCTA A (SEQ ID NO: 68)

TABLE 27 Alignment of ‘TesA sequences ‘TesA--MADTLLILGDSLSAGYRMSASAAWPALLNDKWQSKT---SVVNASISGDTSQQGLARL 55 PatrA--MADTLLILGDSLSAGYQMPAANAWPTLLNTQWQTQKKGIAVVNASISGDTTAQGLARL PlumA--MADTLLILGDSLSAGYHLPIEQSWPALMEKKWQKSGNKITVINGSISGNTAAQGLERL PproAMAWGNTLLVVGDSLSAGYQMRAEQSWPVLLQPALKQQGHEITVVNASISGDTTGNGLARL VhA--MSEKLLVLGDSLSAGYQMPIEESWPSLLPGALLEHGQDVKVVNGSISGDTTGNGLARL PputA--MAGTLLVVGDSISAGFGLDSRQGWVSLLQARLRDEGFDDKVVNASISGDTSAGGQARL ‘TesAPALLKQHQPRWVLVELGGNDGLRGFQPQQTEQTLRQILQDVKAANAEPLLMQIRLPANYG 115 PatrAPALLKQHQPRWVLIELGGNDGLRGFPAPNIEQDLAKIITLVKQANAKPLLMQVRLPTNYG PlumAPELLKQHKPRWVLIELGANDGLRGFPPQHTEQDLQQIITLVKQANIQPLLMQIRLPPNYG PproAPTLLQQHKPAYVIIELGANDGLRGFPQGTIRNNLSQMITEIQNADAKPMLVQIKVPPNYG VhAPSLLEQHTPDLVLIELGANDGLRGFPPKLITLNLSKMITMIKDSGADVVMMQIRVPPNYG PputAPALLAAHKPSLVVLELGGNDGLRGQPPAQLQQNLASMIERSRQAGAKVLLLGMRLPPNYG ‘TesARRYNEAFSAIYPKLAKEFDVPLLPFFMEEVYLKPQWMQDDGIHPNRDAQPFIADWMAKQL 175 PatrARRYTESFSNIYPKLAEQFALPLLPFFMEQVYLKPEWIMEDGIHPTRDAQPFIAEWMAKQL PlumARRYTESFAKIYPKLAEYNQIPLLPFYMEQVAIKPEWVQQDGLHPNLAAQPFIADWMSDTL PproAKRYSDMFSSIYPQLSKELATPLLPFFLEQIILKQEWMMNDGLHPKSDAQPWIAEYMAENI VhAKRYSDMFYDIYPKLAEHQQVALMPFFLEHVIIKPEWMMDDGLHPKPEAQPYIADFVAQEL PputAVRYTTAFAKVYEQLAADKQVPLVPFFLEGVGGVPELMQADGIHPAQGAQQRLLENAWPAI ‘TesAQPLVNHDS 183 PatrA EPLVNHES PlumA SAHLNYS PproA APYL VhA VKHL PputAKPLL.

Example 37. Production of Fame in the Absence of a Wax Synthase inFermentors

This Example demonstrates that a process as described in Example 36,supra, can be scaled up to produce fatty acid esters at commercial scalein accordance with the present invention.

Cells from a frozen stock were revived in an LB broth for 4-8 hours andthen cultured in a defined medium containing: 1.5 g/L of KH₂PO₄, 4.54g/L of K₂HPO₄ trihydrate, 4 g/L of (NH₄)₂SO₄, 0.15 g/L of MgSO₄heptahydrate, 20 g/L of glucose, 200 mM of Bis-Tris buffer (pH 7.2),1.25, and 1.25 mL/L of a vitamin solution. The trace metals solutioncomprised 27 g/L of FeCl₃.6H₂O, 2 g/L of ZnCl₂.4H₂O, 2 g/L ofCaCl₂.6H₂O, 2 g/L of Na₂MoO₄.2H₂O, 1.9 g/L of CuSO₄.5H₂O, 0.5 g/L ofH₃BO₃, and 100 mL/L of concentrated HCl. The vitamin solution comprised0.42 g/L of riboflavin, 5.4 g/L of pantothenic acid, 6 g/L of niacin,1.4 g/L of pyridoxine, 0.06 g/L of biotin, and 0.04 g/L of folic acid.

100 mL of a culture grown overnight was used to inoculate 2 liters ofthe same medium, but with only 2 g/L of glucose, in a fermentor undertightly controlled temperature, pH, agitation, aeration and dissolvedoxygen. The conditions in the fermentor were 32° C., pH 6.8, and adissolved oxygen (DO) level equal to 30% of saturation. The pH wasmaintained by addition of NH₄OH, which also acted as a nitrogen sourcefor cell growth. When the initial glucose became almost consumed, a feedcontaining 60% glucose, 3.9 g/L MgSO₄ heptahydrate and 10 mL/L of thetrace minerals solution was supplied to the fermentor. The feed rate wasset up to match the cell growth rate to avoid accumulation of glucose inthe fermentor. By avoiding glucose accumulation, it was possible toreduce or eliminate the formation of byproducts such as acetate, formateand ethanol, which are otherwise commonly produced by E. coli. Duringthe first 16-24 hours, the feed was supplied exponentially, allowing thecells to grow at a fixed growth rate. Once the feed rate reached adesired maximum (from 6 to 10 g glucose/L fermentor/h) it was maintainedat that level for the remainder of the fermentation run. In the earlyphases of the growth, the production of FAME was induced by the additionof 1 mM IPTG and 25 mL/L of pure methanol. The fermentation was allowedto continue for a period of 3 days. Methanol was added several timesduring the run to replenish what had been consumed by the cells, butmostly what had been lost by evaporation in the off-gas. The additionswere used to maintain the concentration of methanol in the fermentationbroth at between 10 and 30 mL/L, so as to guarantee efficient productionwhile avoiding inhibition of cell growth.

The progression of the fermentation was followed by measurements ofOD600 (optical density at 600 nm), glucose consumption, and esterproduction.

Glucose consumption throughout the fermentation was analyzed by HighPressure Liquid Chromatography (HPLC). The HPLC analysis was performedaccording to methods commonly used for certain sugars and organic acidsin the art, using, for example, the following conditions: Agilent HPLC1200 Series with Refractive Index detector; Column: Aminex HPX-87H, 300mm×7.8 mm; column temperature: 35° C.; mobile phase: 0.01 M H₂SO₄(aqueous); flow rate: 0.6 mL/min; injection volume: 20 μl.

The production of fatty acid methyl and ethyl esters was analyzed by gaschromatography with a flame ionization detector (GC-FID). The samplesfrom fermentation broth were extracted with ethyl acetate in a ratio of1:1 vol/vol. After strong vortexing, the samples were centrifuged andthe organic phase was analyzed by gas chromatography (GC). The analysisconditions were as follows:

Instrument: Trace GC Ultra, Thermo Electron Corporation with Flameionization detector (FID) detector;

Column: DB-1 (1% diphenyl siloxane; 99% dimethyl siloxane) CO1 UFM1/0.1/5 01 DET from Thermo Electron Corporation, phase pH 5, FT: 0.4 μm,length 5m, id: 0.1 mm;

Inlet conditions: 250° C. splitless, 3.8 minute 1/25 split method useddepending upon sample concentration with split flow of 75 mL/min;

Carrier gas, flow rate: Helium, 3.0 mL/min;

Block temperature: 330° C.;

Oven temperature: 0.5 minute hold at 50° C.; 100° C./minute to 330° C.;0.5 minute hold at 330° C.;

Detector temperature: 300° C.;

Injection volume: 2 μL;

Run time/flow rate: 6.3 min/3.0 mL/min (splitless method), 3.8 min/1.5mL/min (split 1/25 method), 3.04 min/1.2 mL/min (split 1/50 method).

This protocol was applied in fermentation runs of two different strains:ID1 (MG1655 ΔfadE::PTRc tesA-fadD) and IDG5 (MG1655 ΔfadE ΔfhuA ΔadhΔldh ΔpflB::P_(TRC) tesA, P_(T51)fadD), neither of which contained thegene coding for an ester synthase. Cells were induced at 4 hours afterinoculation by an IPTG addition, and methanol was fed to the fermentersto provide the alcohol for production of FAMEs. In separate experiments,the cultures were fed glucose at two different maximum feed rates: 6 and10 g/L/h.

With both strains and at each glucose feed rate, the cultures indicateda preference for the production of FAME over free fatty acids, as shownin FIG. 63 and FIG. 64. In 70-hour fermentations, ID1 produced about 19g/L of FAME and less than 1 g/L FFA when fed at 6 g/L/h, and produced 28g/L FAME and about 1 g/L FFA when fed at 10 g/L/h. IDG5 produced 20 g/LFAME and less than 1 g/L FFA at the lower glucose feed, and produced 25g/L FAME and about 10 g/L FFA at the higher glucose feed.

Example 38. Identification of Naturally-Occurring Thioesterases forAltered Properties Based on Protein Engineering Results

E. coli 'TesA engineering experiments conducted herein are useful inidentifying many amino acid residues, the mutations of which lead toaltered properties. 'TesA is an enzyme that belongs to the SGNH family,a broad category of enzymes. It is likely that other homologs of 'TesAcan also be used in the production of biodiesel using the pathwaysdescribed herein. This example identifies homologs of 'TesA withpotentially altered properties as compared to 'TesA. The method isoutlined below.

Homologs of 'TesA were identified using the strategy outlined below.

The homologs with substitutions at the positions corresponding to thoseidentified in the 'TesA screen are shown in FIG. 55. The homolog ID andthe sequence alignment near the positions of interest are also shown.

EQUIVALENTS

While specific examples of the subject inventions are explicitlydisclosed herein, the above specification and examples herein areillustrative and not restrictive. Many variations of the inventions willbecome apparent to those skilled in the art upon review of thisspecification including the examples. The full scope of the inventionsshould be determined by reference to the examples, along with their fullscope of equivalents, and the specification, along with such variations.

All publications, patents, patent applications, and other referencescited in this application are herein incorporated by reference in theirentirety as if each publication, patent, patent application or otherreference were specifically and individually indicated to beincorporated by reference.

1.-36. (canceled)
 37. A recombinant cell comprising a mutantthioesterase having an amino acid sequence at least 90% identical to SEQID NO: 73 and having a substitution at an amino acid position selectedfrom the group consisting of position 13, 19, 37, 39, 43, 44, 47, 76,78, 87, 95, 104, 108, 132, 145, 158, 163, and 164, wherein the mutantthioesterase converts fatty acyl substrates to fatty esters and hasincreased substrate specificity for C10, C12, C14 substrates relative toSEQ ID NO:
 73. 38. The recombinant cell of claim 37, further comprisinga heterologous carboxylic acid reductase enzyme and an alcoholdehydrogenase enzyme.
 39. The recombinant cell of claim 38, wherein theheterologous carboxylic acid reductase enzyme is a member selected fromthe group consisting of fadD9, carA and carB.
 40. The recombinant cellof claim 39, wherein the carboxylic acid enzyme is carB.
 41. A cellculture comprising the recombinant cell of claim
 37. 42. A method forpreparing C10, C12 or C14 fatty acid derivatives, the method comprising:cultivating a recombinant cell that comprises: an engineeredthioesterase enzyme that has an increased substrate specificity for C10,C12, C14 substrates, wherein the engineered thioesterase enzyme has anamino acid sequence that is at least 70% identical to SEQ ID NO: 73 andhas at least one substitution mutation at an amino acid positionselected from the group consisting of position 19, 43, 78, 95, 108, 132and
 145. 43. The method of claim 42, wherein the fatty acid derivativeis a fatty acid.
 44. The method of claim 43, wherein the fatty acidderivative is a C12 fatty acid.
 45. The method of claim 44, wherein therecombinant cell further comprises a heterologous carboxylic acidreductase enzyme and an alcohol dehydrogenase enzyme and the fatty acidderivative is a fatty alcohol.
 46. The method of claim 45, wherein theheterologous carboxylic acid reductase is carB.
 47. The method of claim45, wherein the fatty alcohol is a C12 alcohol.